High Throughput Functional Genomics

ABSTRACT

This invention focuses on the marriage of solid-state electronics and neuronal function to create a new high-throughput electrophysiological assay to determine a compound&#39;s acute and chronic effect on cellular function. Electronics, surface chemistry, biotechnology, and fundamental neuroscience are integrated to provide an assay where the reporter element is an array of electrically active cells. This innovative technology can be applied to neurotoxicity, and to screening compounds from combinatorial chemistry, gene function analysis, and basic neuroscience applications. The system of the invention analyzes how the action potential is interrupted by drugs or toxins. Differences in the action potentials are due to individual toxins acting on different biochemical pathways, which in turn affects different ion channels, thereby changing the peak shape of the action potential differently for each toxin. Algorithms to analyze the action potential peak shape differences are used to indicate the pathway(s) affected by the presence of a new drug or compound; from that, aspects of its function in that cell are deduced. This observation can be exploited to determine the functional category of biochemical action of an unknown compound. An important aspect of the invention is surface chemistry that permits establishment of a high impedance seal between cell and a metal microelectrode. This seal recreates the interface that enables functional patch-clamp electrophysiology with glass micropipettes, and allows extracellular electrophysiology on a microelectrode array. Thus, the invention teaches the feasibility of using living cells as diagnostics for high throughput real-time assays of cell function.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a non-provisional application that claims priorityto U.S. Provisional Application No. 60/135,275, filed May 21, 1999, thecomplete disclosure of which is incorporated by reference herein.

1. FIELD OF THE INVENTION

The present invention relates to an apparatus, processes, methods andsystems for the analysis of samples and test substances, includingdrags, toxins, genes, gene products and the like. Moreover, the presentinvention discloses high throughput methods particularly suitable forconducting functional genomics studies by which information about thefunction of isolated nucleic acids can be obtained without resorting tocumbersome conventional methods like the creation of transgenic animalsor animals in which one or more selected genes have been “knocked out.”In particular, the present invention makes possible the gathering of thetypes of information about the physiological, pharmacological, or otherbiological effects of a sample or a test substance under conditions thatmimic in vivo studies.

2. BACKGROUND OF THE INVENTION

At the present time, the only available “assays” based on cognitive or“cellular function” are living creatures. The use of C. elegans andDrosophila is predominant in these assays. In vitro cell cultures ofembryonic rat and mouse tissue, especially through “knockout”technology, have also been used to study cellular organization andcommunication. However, in studies to date, single neurons have not beenelectrically integrated with modern electronics except in expensivepatch-clamp methodology, which can be tedious, can result indisorganized cultures and are incapable of high throughput analysis.Imaging capabilities have been introduced using voltage-sensitive dyes.Such techniques are limited in their use, however, and dyes aregenerally toxic to neurons, as already mentioned above.

Therefore, it is clear that there is a lack of in vitro assays forstudying neurotoxicity, for example, which are based on a cell'sfunction. There are also very few methods of measuring toxicity otherthan morphological analysis. There is also a problem performing chronicelectrophysiological monitoring of cells by standard electrophysiology.What is more the field of genomics lacks a high throughput assay toanalyze the large number of sequences and genes being uncovered by thehuman genome project. Thousands upon thousands of new compounds are alsobeing generated by recently utilized combinatorial chemical synthesismethods.

The use of biological cells and their underlying cellular functions asmodel systems for information processing is being investigated for anumber of practical applications. Algorithms that are based on olfactoryprocessing (Ambrose-Ingerson, J., Granger, R., and Lynch, G. (1990).Science 247: 1344-1348.; Granger, R., Ambrose-Ingerson, J., Anton, P.S., Whitson, J. and Lynch, G. (1991). An Introduction to Neural andElectronic Networks, eds. Zornetzer, S. F., Davis, J. L., and Lau, C.(Academic Press, Inc., San Diego), pp. 25-42.), computing using DNA in atest tube (Adleman, L. (1994). Molecular Computation of Solutions toCombinatorial Problems. Science Vol. 266.), or possibly manipulation ofDNA in bacteria or other cells are just some examples. Recent articleshave focused on bioinformation and the creation of biological pathwaysor genetic circuits using silicon-based models (Palsson, 1997).Experiments on tissue slice preparations, in cultured neuronal networks(Gross. G. W., Rhoades, B. K., Azzazy, H. M. E., & Wu, M. C., (1995).The use of neuronal networks on multielectrode arrays as biosensors.Biosens. Bioelectron., 10, 553-567.; 1: Stenger D A, Hickman J J,Bateman K E, Ravenscroft M S, Ma W, Pancrazio J J, Shaffer K, SchaffnerA E, Cribbs D H, Cotman C W. Related Articles Microlithographicdetermination of axonal/dendritic polarity in cultured hippocampalneurons J Neurosci Methods. 1998 Aug. 1; 82(2): 167-73.) and with singleneurons (e.g. LeMasson, G., E. Marder and L. F. Abbott (1993)Activity-dependent regulation of conductances in model neurons. Science259, 1915-7.; Marder, E. and L. F. Abbott (1995) Theory in motion. CurrOpin Neurobiol 5, 832-40.; Schizas, C. N. and C. S. Pattichis (1997)Learning systems in biosignal analysis. Biosystems 41, 105-25.) arebeing studied using dual patch-clamp electrophysiology and imagingsystems. Many others have proposed “in silica” models of intracellularfunction as precursors to programming cells for biological computation.See, e.g., U.S. Pat. No. 5,648,926 to Douglas et al., the contents ofwhich are incorporated herein by reference. This application describes ahybrid system to elucidate cellular information in an efficient manner.There is a need to combine new algorithms to reproduce physiologicalconditions found in human, or modeled using data obtained with C.elegans and Drosophila that combines speed and utility of siliconsystems and the relevance of cellular physiology to create newbiological/non-biological high throughput assays.

With the increased capacity to uncover, isolate, discover, or create newsubstances (or even finding new uses for old substances), including newgenetic materials, the need for a “functional assay,” which providessome idea of the potential effects, roles, or functions of the testsubstance, is even more acute. One can look at function from thestandpoint of what is the function or role of molecules, compounds andproteins in an organism. One can also look at function from thestandpoint of how collections of molecules, compounds and proteinscreate or affect pathways that are combined to comprise functionalcategories in a cell such as energy metabolism, extracellular signaling,transcription, or protein synthesis. These pathways underlie theprocesses and functions that maintain a cell and their identificationhelps to establish the identity and role or “cellular function” of thelest substance in a higher organism. Particular groups of cells havingunique “cellular functions” are intermixed in an organized fashion tocreate a higher organism such as an animal.

Traditionally, it has been difficult to assay the effect of a compoundor protein on a cell's internal “functional categories” withoutobserving the whole organism over a period of lime. Many assays havebeen developed to gain information before resorting to experiments atthe whole organism level, with mixed results, In vitro biochemicalassays attempt to reproduced some of these pathways outside the cell,but such assays lack the interactions with the myriad of other pathwaysin the cell.

Fluorescence probes, microsensors and electrophysiological recordingshave supplied a wealth of information but suffer from many drawbacks.Patch-clamp electrophysiological recordings can provide acutemeasurements but the experimental conditions lead to cell death. Thisdrawback also applies to most fluorescent probes, which can cause toxiceffects through photobleaching. Thus, a need exists in drug discovery,functional genomics and basic science for an assay that providesinformation and data about molecules, compounds and proteins (or theirgenes) as these substances interact non-invasively with a living celland its various cellular pathways or functional categories over a periodof time. Preferably, information and data about the various cellularpathways or functional categories, which are affected, are obtained fromsuch an assay. More preferably, it would be desirable if suchinformation and data could be captured electrophysiologically. If onecan combine such features with simple sample preparation and if such anassay allows many conditions to be tested quickly on a statisticallyrelevant number of cells, then one will have provided a very usefulassay having a high throughput cellular functional capacity and whichcan provide much of the information that formerly could only be obtainedthrough experiments at the whole organism level or by extensive and timeconsuming experimentation. A wide variety of biological compounds affectcellular functions. Marder has shown that greater than 40 biochemicalsare involved in just the communication between neurons in the lobsterdigestive system. (Marder, E. and L. F. Abbott (1995) Theory in motion.Curr Opin Neurobiol 5, 832-40.) Many of these biochemicals are specificfor ion channels, but many more act through receptors. Similarinformation for other systems about the interaction of knownbiochemicals and cellular processes or pathways can also be gleened fromany neuroscience text. In addition, there is a wealth of clinical andepidemiological data that shows how a whole host of compounds affectcellular function, for example.

By indirect reference, the applicant believes that a large number ofthese biochemicals must affect the membrane potential of an affectedcell in some way. For example, some compounds (such as saxitoxin)operate by inhibition of the sodium ion channel. Others, such astetraethylammonium chloride (TEA) operate by acting on the potassiumchannel. Still other compounds activate intracellular cascades leadingto calcium mobilization and specific gene activation. Hence, theapplicant describes herein, systems, devices and methods that exploitthe effects of biochemicals on inter alia the membrane potential.Accordingly, the applicant has discovered that one can characterize thechanges in an action potential obtained from an electrically active cellfollowing the addition of specific biochemical compounds or “triggers”to such electrically active cells (e.g., neuronal cells) using planermicroelectrodes that enables elucidation of the cellular functionrelevant to drug discovery or functional genomics. It should be notedthat a particular embodiment of the changes in a membrane potential arethe changes that can be observed in an action potential. Hence, anelectrically active cell is one that exhibits perceptible (measurable)changes in its membrane potential, more preferably, one that exhibitsperceptible changes in its action potential.

Examples of biochemicals of interest include, but are not limited to,those that elicit changes in signals via the following mechanisms: (a)phosphatidylinositol turn-over; (b) calcium mobilization; (c)phosphorylation of intracellular protein messengers; (d) ion channelblockers (Na⁺, K⁺, Ca²⁺, etc.); and (c) cAMP formation. Biochemicals canalso be selected for their inhibitory properties on specific pathways,such as neurotransmission inhibitors and protein synthesis inhibitors.Some of these compounds have been shown to affect the membrane potentialand other individual ion channels.

Surface modification technology utilizing Self Assembled Monolayers(SAMs) is a known process for preparing a modifying layer composed oforganic molecules, which can spontaneously form strong interactions orcovalent bonds with reactive groups on an exposed surface. Theutilization of SAMs for modifying surfaces has been demonstrated onelectronic materials such as silicon dioxide, biodegradable polymers andoilier polymers such as Teflon. A large variety of functional groups orcombination of functional groups can be located on the terminus oppositethe attachment point of a SAM, and the chemical composition can bemanipulated to systematically vary the surface free energy. Biologicalcells can attach to, and proliferate on SAMs, and SAMs can be used topattern a surface. SAMs are also useful to set as templates for thepatterning of biomolecules, especially antibodies. SAMs thus can proveto be an ideal tool for the design of artificial surfaces for thetailoring of cellular interactions.

Metal microelectrodes surrounded by an insulator can be used to recordthe electrical activity of cells extracellularly. The applicant hassurmised that if the interface can be tailored to keep the cells on themicroelectrodes, a viable system can be created for high throughput cellassays. The applicant believes that this type of system can befabricated by taking advantage of previous work involving orthogonalself-assembly on two different metals (sec, e.g., Hickman, J. J.Laibinis, P. E., Auerbach, D. I., Zou, C, Gardner, T. J., Whitesides, G.M., and Wrighton, M. S. (1992). Toward orthogonal self-assembly of redoxactive molecules on Pi and Au: Selective reaction of disulfide with Auand isonitrile with Pi. Langmuir 8: 357.) and on a surface composed of ametal and an insulator coating region. See, also, U.S. Pat. No.5,223,117 to Wrighton et al., the contents of which are incorporatedherein by reference.

Surface analysis techniques have been applied to analyze cell culturesurfaces both before and after culture and to relate the quantitativeand qualitative results to cell morphology and survival. (See, e.g.,Schaffner, A., Barker, J. L., Stenger, D. A., and Hickman. J. (1995).Investigation of the factors necessary for growth of hippocampal neuronsin a defined system. J. Neurosci. Methods, 62, 111-119.) Previousstudies by others have also correlated cell behavior to the initiallyquantified properties of the culture surface, i.e., prior to theaddition of cells. Many components of the culture medium adsorb onto thesurface, and cells secrete substances that comprise an extracellularmatrix (ECM), as well as soluble molecules. Many of these biomoleculespotentially are the source of the cell behavior monitored and can be avaluable source of information.

One problem encountered using a cell line as the sensor element is thatcell lines (e.g., NG108-15, which is derived from aglioma×neuroblastoma) have an inherently unstable genome. The applicantconsiders primary cells to be very relevant to the present systembecause it is presumed that such cells more closely approximate in vivosystems than tumor-derived cell lines; however, primary cells tend to bedifficult to culture and are in homogeneous. A possible solution tothese drawbacks involves the utilization of clonal cell lines derivedfrom CNS stem cells. Thus, a preferred cell having a stable long-livedphenotype is one derived from a stem cell. In the present invention,each individual cell becomes a unique assay element with the cellslocalized on individual microelectrodes. Statistics can be performed ona reproducible population in response to a compound that is introducedinto the media. Further we will apply system level algorithms to enablethe reproduction or representation of relevant physiological states orreproductions of known assays employed by pharmaceutical or otherbiotechnology companies.

Hence, the present invention hopes to provide an assay of cellularfunction, using “functional categories” within the cell as defined, forexample, by Riley, M. (1993). Functions of gene products of Escherichiacoli. Microbiol. Rev. 57, 862-952. The present system is validated bytaking known biochemicals with known functions and monitoring thechanges in electrical potential upon introduction of the knownbiochemicals in the media. The present invention and its broadlyapplicable techniques would add a new paradigm in molecular functionanalysis, including gene function analysis. It is also possible to mapcells in varying stages of development, as the present techniques can beapplied using embryonic cells. Particularly useful cells include CNScells, but the present approach can be used on any cell type thatpermits the monitoring of electrical changes in the membrane potential.It is hoped that a clear need for the present invention has beenestablished by the discussion presented herein.

3. SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to systems, devices,processes and methods for determining the effects of test substances,including their physiological or pharmacological effects on cells.Convenient measures of the effects of test substances are the effects onthe electrical activity of cells, which electrical activity can bemanifested by cells in a number of ways, including changes in membranepotential. In particular, one can measure changes in a cell's actionpotential, which can be considered an expression of the chances inmembrane potential over a given lime period. Thus, the present applicanthas discovered that the effects of test substances can be studied andrevealed by recording and examining the changes in the electricalcharacteristics of electrically active cells, which changes are areflection of the effects of the test substance on the physiology of thecell, including effects on underlying cellular processes, mechanisms, orpathways. The present invention, then, can serve as an important measureof the biological activity of a test substance or, at least, anindicator of potential biological activity of a test substance incertain categories of cellular function. Such measurements orindications are conveniently made available without resorting to the useof in vivo models and represent a significant advance in the art.

In particular, the present invention seeks to provide systems andmethods for the deconvolution of an action potential recorded from anelectrically active cell, which cell is positioned on the surface of asolid state microelectrode. More particularly, a cell is exposed to avariety of conditions, and the effects of those conditions, or changesin such conditions, on the observed action potential are noted. Makinguse of the knowledge accumulated about the physiological,pharmacological and related effects (e.g., mechanistic pathwayselucidated in the literature) of known substances, the present inventionmakes possible the further elucidation of the changes in one or morecharacteristics of the electrical activity of a cell (as reflected, forexample, in its action potential), which changes can thus be associatedor correlated with the specific effect or mechanistic pathway identifiedwith each substance or combinations thereof. Hence, in a specificembodiment of the present invention, a body of knowledge is providedwhich permits the examination of test substances to determine theireffects on the action potential and, in turn, on the underlyingprocesses or functional categories of the cell affected by the testsubstances.

In a specific embodiment of the invention, a system is provided, whichis capable of identifying one or more ion channels of a cell, whichchannels are affected by a test substance. Such a system comprises adevice, which is optionally accompanied by software (e.g., a computerprogram, data processing application, algorithm and the like), in whichthe device comprises: (a) a solid state microelectrode; (b) a cellculture comprising one or more electrically active cells having a cellmembrane including one or more ion channels, which one or more cells arecapable of providing a measurable change in their electricalcharacteristics (for example, provides a measurable action potentialthat exhibits one or more perceptible characteristics); (c) anintervening layer which (i) comprises a surface modifying agent, and(ii) is positioned between the microelectrode and the one or more cellsof the cell culture, such that a high impedance seal is provided atleast in the vicinity of the one or more cells of the cell culture. Theoptional accompanying software comprises instructions that can beimplemented by a computer and which are capable of relating changes inthe one or more characteristics exhibited by the electrical activity(e.g., exhibited by the action potential) to one or more ion channels ofthe one or more cells upon exposure of the one or more cells to a lestsubstance. More particularly, the applicant conceives of a highimpedance seal that reduces the lateral flow of ions across themicroelectrode from the surrounding medium, while permitting orfacilitating the vertical flow of ions between the cell and themicroelectrode. In this manner, the microelectrode is best suited todetect changes in the ion flux attributable to the cell and not due tothe surrounding medium.

The present invention contemplates a system in which the one or morecharacteristics exhibited by the membrane potential or action potentialis manifested in its waveform or a derivative thereof. In still otherembodiments, the one or more characteristics include at least one ofafter potential, time to cessation of activity, frequency, amplitude,shape, spike rate, or time constant. In preferred embodiments of thepresent invention, the data processing instructions are further capableof receiving input data comprising data on the temporal description ofthe membrane potential, action potential, or the changes therein.

In still additional embodiments, a system is provided in which dataprocessing instructions are further capable of receiving input datacomprising data on ion flux through ion channels selected from the groupconsisting of sodium channels, potassium channels, calcium channels, orcombinations thereof. Other aspects of a system of the invention includeutilization of a planar microelectrode in which the microelectrode canbe a field effect transducer (FET). It is further contemplated that thesystem further comprises an insulator that surrounds the metalmicroelectrode or covers the gate of the FET. A suitable insulatorincludes materials selected from, but not limited to, the groupconsisting of silicon, modified silicon dioxide, silicon nitride,silicon carbide, germanium, silica, gallium, arsenide, epoxy resin,polystyrene, polysulfone, alumina, silicone, fluoropolymer, polyester,acrylic copolymers, polylactate, or combinations thereof.

A suitable cell culture for use in the present invention comprises anelectrically active cell, which can include any metabolically activecell. Examples include, but are not limited to a neuronal cell or acardiac cell. Preferably, the system makes use of a NG-108 cell. Stillother cell cultures comprise a hippocampal cell, a stem cell, atransformed stem cell, their respective progeny, or combinationsthereof. Moreover, one can contemplate the use of a stem cell or otherprogenitor or precursor cell, which has been exposed to adifferentiating factor.

The present invention includes the use of a surface modifying agent,preferably comprising a self-assembling monolayer. Examples of suitablesurface modifying agents include, but are not limited to, silanes,thiols, isocyanides, polyelectrolytes and the like, or combinationsthereof. More preferably, the system incorporates an intervening layerthat further comprises cell anchorage molecules. Suitable cell anchoragemolecules include, but are not limited to, antibodies, antigens,receptor ligands, receptors, lectins, carbohydrates, enzymes, enzymeinhibitors, biotin, avidin, streptavidin, cadherins. RGD-type peptides,integrins, cadherins, modified lipids, or combinations thereof.

In a specific embodiment of the present invention the intervening layercomprises a high viscosity mixture comprising alcohols, ethers, esters,ketones, amides, glycols, amino acids, saccharides,carboxymethylsaccharides, carboxyethylsaccharides, aminosaccharides,acetylaminosaccharides, polymers thereof, or combinations thereof. In apreferred embodiment of the invention, the cell culture is coated with apolymer, such as cellulose, methylcellulose, dextran and the like. Stillother features of a preferred embodiment of the invention include anintervening layer that can be characterized as either an attractivelayer or a repulsive layer. The preferred system further comprises adetector circuit. One might also use cells transfected with endogenousor exogenous nucleic acid as the one or more cells of the cell culture.In such a case, the nucleic acid can comprise a nucleotide sequenceassociated with known or unknown function.

It is also an object of the invention to provide a method of determiningone or more ion channels that are affected by a test substancecomprising: (a) contacting a substance to be tested with a devicecomprising a solid state microelectrode; a cell culture including one ormore cells having a cell membrane including one or more ion channels,which one or more cells are capable of providing a measurable actionpotential that exhibits one or more perceptible characteristics; and anintervening layer that is acting as a high impedance seal and which ispositioned between the microelectrode and the cell culture; (b)collecting data on the action potential, the one or more characteristicsthereof, or one or more changes therein; and (c) determining from thedata the one or more ion channels that are affected by the testsubstance. In specific embodiments of the present invention, the testsubstance comprises a toxin, a drug, a pathogen, a neurotransmitter, anerve agent, or mixtures thereof. Preferably, the method utilizes adetermining step that includes deconvoluting the action potential, theone or more characteristics thereof, or the one or more changes therein.

The present invention is also directed to a system by which one can usealgorithms that mimic physiological states or conditions of interest togauge or determine the benefits, effects, side effects, or unintendedconsequences, etc. of test substances under such test conditions. Forexample, the system contemplated by the present invention can bemanipulated so that particular pathways are turned on or off or areisolated in a way that provides the best scenario for observing theresulting behavior of a cell (e.g., a neuronal cell) upon exposure toone or more given test substances. As another example, one might beinterested to know what the effects of a candidate drug for depressionmight have on individuals having high blood sugar levels e.g., diabeticsor simply people who have just consumed a high carbohydrate meal or whoare on a certain diet), or low blood sugar levels (e.g., hypoglycemics),or on individuals with high cholesterol. The system of the presentinvention can then be adjusted (where the adjustment of the variables ofthe system are controlled and kept track of by a system computer programor other accompanying system software), for example, by the addition orremoval of cell culture nutrients, the transfection of the cells of thecell culture with a given nucleic acid or exposing them to a protein,peptide, or small molecule, changing the chemical or electrochemicalcharacteristics of the media to the cell culture and the like. In thismanner, the system of the present invention is able to provideinformation on physiological or pharmacological effects of drugcandidates, which were previously available only from animal or humanstudies.

Accordingly, in another aspect of the present invention, a system iscontemplated having a high throughput capacity to determine potentialphysiological effects of a test substance comprising a device andaccompanying software, in which the device comprises a solid statemicroelectrode and a cell culture comprising one or more cells thatexhibit electrical activity or, preferably, which are capable ofproviding a measurable action potential that exhibits one or moreperceptible characteristics. The system can also optionally compriseaccompanying software, which itself comprises data processinginstructions capable of relating changes in the electrical activity or,preferably, in the one or more characteristics exhibited by the actionpotential to one or more potential physiological effects exerted by atest substance upon exposure of the test substance to the one or morecells of the cell culture. In particular, such a device may furthercomprise an intervening layer that is acting as a high impedance sealand which is positioned between the microelectrode and the one or morecells of the cell culture, and in which the accompanying softwarefurther comprises instructions, which can be implemented by a computer,for manipulating one or more system parameters to alter one or moreconditions of a given experiment, for interpreting the outcome of suchmanipulations, or for both. Of course, separate software programs can bewritten to divide specific tasks, as dictated by system requirements,design, or convenience.

The present invention contemplates manipulations that include theaddition of a compound of interest to the cell culture or the removalthereof from the cell culture. In particular, the compound of interestmight be a nutritive material or a cell modulator, and the dataprocessing instructions may include a temporal analysis of the actionpotential or the changes observed therein. More specifically, the dataprocessing instructions is capable of providing an output suggestive ofthe involvement of one or more cellular pathways or receptors ofinterest. The present system preferably may be accompanied by softwarethat includes instructions for a feedback loop to provide forflexibility in the manipulation of the parameters of the system toenhance or maximize the desired outcomes.

An object of the present invention is the realization of a system thatis capable of determining a mode of action of a test substance based onthe one or more cellular pathways or receptors of interest involved.

In a system of the present invention, the test substance might comprisea toxin, a drug candidate, a pathogenic agent, a neurotransmitter, anerve agent, a gene, a gene product, or mixtures thereof. Consistentwith the objectives of the present invention, a system is provided fordetermining one or more potential functions of an isolated nucleic acid,its expression product, or one or more active fragments of the nucleicacid or expression product, comprising a device and accompanyingsoftware, in which the device comprises a solid state microelectrode anda cell culture comprising one or more cells that are capable ofproviding a measurable action potential that exhibits one or moreperceptible characteristics and which cells have been either transfectedwith an isolated nucleic acid or exposed to its expression product, andin which the accompanying software comprises data processinginstructions capable of relating changes in the one or morecharacteristics exhibited by the action potential to one or morepotential functions of the isolated nucleic acid, its expressionproduct, or one or more active fragments of the nucleic acid orexpression product.

Yet another object of the present invention involves a method ofdetermining one or more potential functions of an isolated nucleic acid,its expression product, or one or more active fragments of the nucleicacid or expression product comprising (a) providing a device comprisinga solid state microelectrode; a cell culture comprising one or morecells that are capable of providing a measurable action potential thatexhibits one or more perceptible characteristics and which cells havebeen either transfected with an isolated nucleic acid or exposed to itsexpression product, (b) collecting data on the action potential, the oneor more characteristics thereof, or one or more changes therein; and (c)determining from the data the one or more potential functions of theisolated nucleic acid, its expression product, or one or more activefragments of the nucleic acid or expression product. As in otherembodiments of the present invention, a preferred determining step isone that includes deconvoluting the action potential, the one or morecharacteristics thereof, or the one or more changes therein; one thatfurther comprises manipulating one or more parameters to alter one ormore conditions of a given experiment; and one that further comprisesinterpreting the outcome of such manipulations.

Still another aspect of the present invention involves a computerreadable medium encoding a program that includes instructions forexecution by a computer, which instructions comprise data processingsteps that relate changes in one or more characteristics exhibited by anobserved action potential to one or more ion channels of one or morecells of a cell culture upon exposure of the one or more cells to a testsubstance. Specifically, the computer readable medium of the presentinvention is one in which the data processing steps may comprise adeconvolution step by which the changes in the one or morecharacteristics exhibited by the observed action potential are comparedwith stored information from past observations allowing the computer toattribute the changes to the one or more ion channels of the one or morecells. It is important to note that in preferred embodiments of thepresent invention, the computer readable medium relating todeconvolution of an action potential (or the deconvolution software) isone in which the data processing steps do not include a spectralanalysis, more particularly, not including a spectral analysis thatmakes use of a Fourier transform. In particular, the deconvolution stepof the present invention is inspired by biological knowledge. That is,our knowledge of the effects or mechanisms by which certain knownsubstances act on the physiology of a cell is utilized by the methods ofthe present invention to more effectively analyze or deconvolute anaction potential to more effectively relate changes in an actionpotential to underlying processes or pathways. It has thus beendiscovered that certain functional categories can be elucidated by thepresent deconvolution step. Hence, at a minimum, one can expose thesystem of the present invention to a test substance and through thedeconvolution process be able to “fit” the effects of test substance onthe action potential recorded by the system to one or more of thesefunctional categories. The determination of the functional categories,in which a test substance best fits, is thus an object of the presentinvention.

Separately, another computer readable medium is provided which permitsthe parameters of the system to be changed and/or manipulated such thatexperimental conditions can be varied. In particular, the instructionsencoded into a preferred computer readable medium are capable ofcustomizing the system to provide desired outcomes on exposure of thesystem to one or more test substances. Such a “system software” may makeof other programs, including deconvolution software. It is important tonote that while the present invention's deconvolution softwarepreferably excludes spectral analysis, more specifically, a Fouriertransformation, the present invention's system software may utilize suchspectral analysis or Fourier transformation.

Yet another object of the present invention is to disclose a systemcomprising: a solid state microelectrode; a cell culture which exhibitsa measurable action potential; an intervening layer comprising a surfacemodifying monolayer, which functions as a high impedance seal; andsoftware capable of analyzing the action potential to elucidate acellular pathway or a receptor of interest. It is a further embodimentof the present invention, a system is disclosed in which the solid statemicroelectrode is a planar microelectrode.

A method is also disclosed, which relates to a high throughput analysis,the method comprising: providing a solid state microelectrode with anintervening layer comprising a surface modifying agent, preferably aself-assembling monolayer, which functions as a high impedance seal;adding a cell culture which exhibits a measurable action potential; andanalyzing the action potential of the cell culture. In a furtherembodiment of the invention, additives are optionally introduced intothe system.

The present invention also contemplates an apparatus comprising: asystem that includes the following elements: a microelectrode; a cellculture which exhibits a measurable action potential; an interveninglayer comprising a surface modifying agent, which functions as a highimpedance seal; and software capable of analyzing the action potentialto elucidate a cellular pathway or a receptor of interest; and a systemalgorithm capable of manipulating the elements of the system to developan assay of interest. An embodiment of the invention is an apparatusthat functions as an assay for determining the mode of action of a drugcandidate on one or more cellular pathways or receptors.

In a specific embodiment of the present invention, a system is providedin which the one or more cells of the cell culture comprise cellstransfected with at least one gene (e.g., one of unknown gene function),and in which the system algorithm is capable of manipulating theelements of the system to assist in the elucidation of effects of thegene on the behavior of the system or for the discovery of genefunction.

Still a further object of the present invention is to provide a methodof detecting an agent comprising: providing an agent; permitting theagent to interact with a sensor, in which the sensor comprises a cellculture having at least one cell which exhibits a measurable membranepotential, a solid state microelectrode, and an intervening layer thatfunctions its a high impedance seal; observing or recording a change inthe membrane potential; and analyzing the change in the membranepotential to elucidate a cellular pathway, ion channel, receptor ofinterest, or the like, which is affected by the action of the agent.

Other objects of the present invention will be apparent to (hose ofordinary skill in the art in view of the discussion and descriptionsprovided herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a illustrates a single extracellular action potential fromspontaneously firing neonatal rat cardiac myocytes cultured for 7 dayson a microelectrode array. FIG. 1 b illustrates a recording from thesame microelectrode site but over a longer time span to demonstrate thepulsatility of the extracellular signal.

FIGS. 2A and 2B illustrates extracellular action potentials from spinalcord neurons on a microelectrode array. The cell firings are initiatedby a depolarizing stimulus.

FIG. 3 illustrates an optical image of neurons cultured on amicroelectrode array.

FIG. 4 illustrates a schematic model of some relevant receptors andintracellular pathways for a NG108-15 cell. Bold face indicates toxinsthat have been tested. The effect of toxins on action potential occurswithin 60-180 seconds, except for VX, which occurs in 15 minutes.

FIG. 5 illustrates results of administration of paraoxon (a nerve agentstimulant with an LD₅₀ of 1.8 mg/kg), ricin and cyanide on NG-108-15cells. The stimulated action potential using intercellular recordingshows the dramatic changes in action potential shape for the duration,amplitude and after hyperpolarization potential (AHP).

FIG. 6 show Hodgkin-Huxley simulations (a—membrane potential;b—derivative of membrane potential) to illustrate sensitivity ofextracellular waveforms to changes in membrane lime constants. Thelargest peak is from a simulation in which the potassium channel timeconstant was lengthened by a factor of five—note the longer afterpotential. The smallest of the peaks results from increasing the sodiumtime constant by a factor of two. The remaining peak is the normal“textbook” Hodgkin-Huxley simulation.

FIG. 7 illustrates a schematic of the neuron-to-microelectrodeinterface, where the capacitive discharge current=(l_(c))=C^(dv/dt).

FIG. 8 illustrates an equivalent circuit for the neuron-microelectrodeinterface, adapted from Fromherz et al., (Fromherz, P., Offenhausser,A., Vetter, T., & Weis, J. (1991). A neuron-silicon junction: A Retziuscell of the leech on an insulated-gate filed-effect transistor. Science252, 1290-1293.)

FIG. 9 illustrates a stacked view of the apparatus, as well as preferredconnectivity diagram for a specific embodiment of a system of thepresent invention.

FIG. 10 provides a flow chart for a deconvolution algorithm.

FIG. 11 provides a flow chart for a system or manipulation algorithm.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The invention has several components that function together fordetermining the effects of a test substance. One embodiment of theinvention is a system comprising a solid state microelectrode; a cellculture which exhibits a electrical activity; and an intervening layer,which functions as a high impedance seal. The system is preferablyaccompanied by software capable of relating changes in the electricalactivity of one or more cells of the cell culture to the physiologicalactivity of the test substance (e.g., capable of deconvoluting an actionpotential). The term “test substance” is meant to cover broadly anysubstance whose effect on a biological system, such as a cell, one isattempting to determine. A test substance includes, but is not limitedto, drugs, proteins, peptides, carbohydrates, nucleic acids, lipids,natural products, small molecules and the like. The “effects” of a testsubstance is likewise broadly construed and may include, but are notlimited to, effects on ion flux, ion channel behavior, underlyingcellular pathways, receptor function and response to agonists orantagonists, gene expression, cause/effect/progression of disease andthe like.

FIG. 9 is a diagram showing the relationship of cells withmicroelectrodes, particularly in a carbon dioxide/molecular oxygenatmosphere. This is a microelectrode array with electrically activecells capable of producing action potentials—a rapid change of voltageacross the membrane of a cell.

Referring to FIG. 9, a liquid or defined medium 4, such as an aqueoussolution of nutrients, surrounds each cell 2. The defined medium 4sustains the viability of each cell 2. The gases in the atmosphere arein equilibrium with dissolved gases in medium 4.

One or more system sensors 6 controls and monitors the parameters of thedefined medium 4. These parameters include pH, temperature andosmolality, for example. Sensors 6 also regulate the nutrient inflowinto the defined medium 4, as well as temperature in response to thesechanges.

In one embodiment, as shown in FIG. 9, a microelectrode 8, in an uppersurface area, is formed with a modified sealed surface 10. Surface 10 isbound to and forms a high impedance seal over the microelectrode 8.Alternatively, and not shown, is an intervening layer that serves toanchor the cell 2. Cell 2 is capable of exhibiting electrical activitythat can be monitored by the microelectrode 8.

Cells are generally separated by a second modified surface 12 (asshown), which is repulsive to cell adherence. Signals from eachmicroelectrode 8 are transmitted by electrode lead wires 14 to adeconvolution algorithm and electronic control system 16. System 16deciphers the role of several ion channels in the action potential ofthe cell 2.

The system algorithm 18 collects information from control system 16 andcreates a database, which relates the properties of the action potentialto different known agents that affect ion channels. The system algorithm18 sends command signals to control system 16. The system algorithm 18also sends commands to the biological system controller 20, whichtransmits updates and status information back to the system algorithm 18and which also controls addition of compounds to test.

5.1. Solid State Microelectrode

In a particular embodiment, the invention comprises a solid statemicroelectrode that can be a flexible or a planar microelectrode. Theflexible microelectrode array comprises a combination of electrodes andinsulator readily adapted to bending, flexing, and twisting to permitpositioning of the microelectrode on a variety of surfaces (or withincertain internal structures). Hence, a microelectrode suitable for thepresent invention is one that can be placed on flat surfaces or oncurved surfaces. The solid state microelectrode of the system can be ametal microelectrode. Moreover, the microelectrode can be a field effecttransducer.

In another particular embodiment of the invention the microelectrode canfurther comprise an insulator and a conductor. Likewise, the conductorof the system can be constructed of gold, platinum, silver, copper,conductive glass, or combinations thereof, or any other suitablematerial. Moreover, the conductive glass of the system can compriseindium tin oxide. Similarly, the active surface of the microelectrode ofthe system can further comprise platinum black or iridium oxide. In oneparticular embodiment of the invention, the insulator surrounding themetal microelectrode of the system can be silicon, modified silicondioxide, silicon nitride, silicon carbide, germanium, silica, gallium,arsenide, epoxy resin, polystyrene, polysulfone, alumina, silicone,fluoropolymer, polyester, acrylic copolymers, polylactate, orcombinations thereof, or other suitable material.

5.2. A Layer Intervening between Microelectrode and Cell

In a particular embodiment of the invention, an intervening layer isestablished between the microelectrode/insulator array and the cellularcomponent. The intervening layer can comprise a surface modifying agent.The surface modifying agent of the system can be a self-assembledmonolayer. In still another embodiment of the system of the invention,the self-assembled monolayer is a silane. Cell anchorage molecules canfurther be used to form part of the intervening layer. In anotherembodiment of the system of the invention the cell anchorage moleculesare antibodies, antigens, receptor ligands, receptors, lectins,carbohydrates, enzymes, enzyme inhibitors, biotin, avidin, streptavidin,cadherins, RGD-type peptides, integrins, cadherins, modified lipids, orcombinations thereof.

In yet still another embodiment of a system of the invention theintervening layer comprises a high viscosity mixture. Suitable highviscosity mixtures include, but are not limited to, alcohols, ethers,esters, ketones, amides, glycols, amino acids, saccharides,carboxymethylsaccharides, carboxyethylsaccharides, aminosaccharides,acetylaminosaccharides, polymers thereof, or combinations thereof.Useful glycols can be polyethylene, polypropylene glycols and the like.In a yet further embodiment of the system of the invention the alcohols,ethers, esters, ketones, amides, glycols, amino acids, saccharides,carboxymethylsaccharides, carboxyethylsaccharides, aminosaccharides,acetylaminosaccharides, polymers (hereof, or combinations thereof areadherent to the monolayer.

The intervening layer of the present invention may further comprise anattractive layer, which increases cell anchorage, or may furthercomprises a repulsive layer, which decreases cell anchorage.Alternatively, the intervening layer can be a pattern of both attractiveand repulsive layers which can confine a cell to a defined area.

5.3. Cells and Media for Cell Culture

The present invention makes use of electrically active cellularcomponents and a variety of cell culture media. The cells of the cellculture are selected from electrically active cells, preferably thosethat give rise to measurable membrane or action potentials. The cellculture of the system can comprise a neuronal cell or a cardiac cellfrom a human, an animal, or an invertebrate. The cell culture cancomprise primary cells or cells of a given cell line. Similarly, theneuronal cell of the system can be a hippocampal cell, a corticalneuron, a cerebellar neuron, a mid-brain neuron, a spinal cord neuron,or a peripheral neuron. Equally well, the cell culture of the system cancomprises a stem cell, the progeny thereof, or a combination thereof. Inone embodiment of the invention, the stem cell of the system can beexposed to a differentiating factor. Transformed (transfected) cells canalso be used in the present cell culture. In a further embodiment of asystem of the invention, the transfected cell comprises a cell harboringa test genomic or cDNA nucleotide sequence. In another embodiment thetest nucleotide sequence can be any gene sequence provided in theGENBANK and updates thereof. Moreover, the test nucleotide sequence canbe any gene sequence provided in the Celera gene database, and updatesthereof.

The cell culture can optionally be coated with a polymer. The polymerthat coats the cell culture can be cellulose, methylcellulose and thelike. Various media can be used in the cell culture of the presentinvention. A preferred medium is serum-free medium. However, usefulmedia can have additives or nutrients to facilitate cell function orgrowth. The additive can include growth factors, vital factors,vitamins, trace elements, and attachment factors. The components of thecell culture can be manipulated. For example, certain substances can beadded, supplemented, or removed depending on the physical orphysiological conditions being mimicked by the cell culture. Hence, anutritive material or a cell modulator can be deleted to provide stressto the cell culture. In another example, glucose can be partly orcompletely deleted from the medium. Glucose or some other carbohydratecan also be added. Additional information on cell culturing techniquescan be found, for example, in Freshney. I. I. “Culture of Animal Cells:A Manual of Basic Techniques.” 4^(th) Ed. Wiley, John & Sons. March2000. The contents of this and all other references provided in thisspecification are incorporated by reference herein.

5.4. Deconvolution Algorithm

In a particular embodiment, the invention comprises at least onealgorithm for deconvolution of the action potential. The deconvolutionanalysis determines the contribution of the several ion channels, ionpumps, and other sources of electrical activity in the formation of theaction potential or action potential train. A detector circuit,preferably a modified Fromherz circuit, can also comprise a system ofthe present invention.

Typically, a deconvolution algorithm is obtained by taking a basalsignal from a system of the present invention; that is one records thesignal (e.g., an action potential) from a base cell culture to which notest substance has been added. Such a basal signal is then stored forlater comparison to a signal recorded from a system in which one of thesystem parameters has been altered or to which a test substance has beenadded. The differences in the recorded signals provides some measure ofthe effects of the altered parameter or added test substance. If certainknown substances have known biological effects are used to obtainrecorded signals, then one eventually can build up a library of changesto the electrical activity, which correlate to the types of biologicaleffects being studied or which are desired. An algorithm is thusobtained from the collective information recorded and stored from theelectrical changes induced by the known substances. This samedeconvolution algorithm is then used to decipher if a test substanceelicits the type of changes in electrical activity, which are indicativeof the biological effects exerted by the known substances. Eventually,knowledge of various functional categories is built up, as illustratedin the Examples Section, below, which serve to indicate the likelihoodthat a given test substance exhibits a physiological effect that can beclassified into one or more of such functional categories.

A more detailed description of the deconvolution algorithm andelectronic control system 16 is provided in FIG. 10. The first stepbegins with a start procedure shown as block S22. The action potentialis then sampled and compared to the database, as shown in block S24. Thenext step requires a determination of whether the action potential thatis measured is within the limits of the parameters of the actionpotential stored in the database, as shown in block S26. If the measuredaction potential falls outside the limits of the parameters of thestored action potential, an error message is displayed (block S28), andthe procedure returns to start.

On the other hand, if the measured action potential falls within thelimits of the parameters of the stored action potential, then one ormore desired compounds are added to the defined medium 4 (block S30).The next step requires a determination of whether the added compoundshave caused a change in the action potential (block S32). If no changesare measured or noted, then the system adds an antagonist substance toblock particular cellular pathways or an agonist substance to stimulateparticular cellular pathways, as needed (block S34).

Here, again, a determination is required as to whether the addedchemical compounds have caused a change in the action potential (blockS36). If no changes are measured or noted, then the cell type or mediumcondition is changed, as at block S37, and the procedure is repeated.

On the other hand, if the chemical compounds added to the defined medium4 have caused a change in the action potential, as at blocks S32 andS36, then a model output action potential is generated (block S38). Themodel output action potential is generally represented by at least threegroups of ions. The first group is sodium, where flux flows from theoutside of the cell to the inside. In the second group of ions, which iscalcium, the flux also flows from the outside of the cell to the inside.In the third group, which is potassium, flux flows from the inside ofthe cell to the outside, and re-establishes the membrane potential.

In block S40, the deconvolution algorithm and electronic control systemdetermines and/or measures the changes in the action potential of themodel output, and compares the measured results to those stored in thedatabase(s) (block S40). The next step requires a determination ofwhether the change in the model output action potential, corresponds toa known functional category of compounds (block S42).

If the change docs not corresponds to a known functional category ofcompounds, then the system adds an antagonist substance to blockparticular cellular pathways or an agonist substance to stimulateparticular cellular pathways, as needed, (block S34), and the stepsproceed as described above.

On the contrary, if the change corresponds to a known functionalcompound category, then an output in the form of a report is presented(block S44).

5.5. Application of a System for Deconvolution on Test Substances

Accordingly, a method is provided, which comprises adding a lestsubstance and analyzing the modified action potential using adeconvolution algorithm. See, FIG. 10. A deconvolving step, in a basicform, involves deciphering the relative contributions of ion fluxesattributable to different ion channels. In a higher form, thedeconvolving step provides information on underlying processes,mechanisms, or pathways that contribute to the changes in the observedion fluxes. In deconvolving the changes in electrical activity (e.g.,changes in the action potential), a waveform, an action potential, or atransformation of same (e.g., a derivative, a frequency function and thelike) may be viewed and analyzed. Conveniently, one can analyze theshape of a wave of an action potential. In a preferred embodiment of thepresent invention, the method is provided, which comprises the use of anoptimized Hodgkin-Huxley waveform.

Other embodiments include temporal analysis of electrical activity,preferably, changes in membrane potential or action potential. Hence,the present invention contemplates the collection of data on, e.g.action potential, the one or more characteristics thereof, or one ormore changes therein, followed by the determination from such data ofwhich one or more ion channels, G-proteins, transporters, ion pumps, orcombinations thereof are affected by the test substance.

5.6. A System for Functional Genomics Analysis

In a specific application of the present invention, a system is providedFor determining one or more potential functions of an isolated nucleicacid, its expression product, or one or more active fragments of suchnucleic acid or expression product. In particular, the system comprisesa device that is optionally accompanied by software. The devicecomprises a solid state microelectrode and a cell culture comprising oneor more cells that are capable of providing a measurable actionpotential that exhibits one or more perceptible characteristics andwhich cells have been either transfected with an isolated nucleic acidor exposed to its expression product. The optional accompanying softwarecomprises data processing instructions capable of relating changes inthe one or more characteristics exhibited by such action potential toone or more potential functions of such isolated nucleic acid, itsexpression product, or one or more active fragments of such nucleic acidor expression product. Preferably, the device further comprises anintervening layer that is acting as a high impedance seal and which ispositioned between the microelectrode and the one or more cells of thecell culture.

Thus, recordings of the electrical signal from transfected cell culturesare recorded and compared to basal recordings. The differences in therecorded and basal electrical signals serve as an indication of theeffects of the nucleic acid used to transform the cells of the cellculture. A high throughput assay for test substances that either mimicthese effects or reverse them can then be undertaken. Alternatively, theeffects of the nucleic acids can be compared to a database of knowneffects and underlying pathways to discern gene function.

5.7. System Algorithm

In one embodiment of the invention, the system comprises at least onealgorithm that analyzes information on changes in ion channel functionto determine which cellular processes are affected. See, FIG. 11.

Referring now to FIG. 11, a more detailed description of the systemalgorithm 18 is provided. This algorithm assures testing of 100compounds or more. The first step begins with a start procedure shown asblock S50. Physiological state parameters, such as polarization values,temperature, etc., are set, as in block S52. Here, an action potentialis generated. The next step requires a determination of whether the setparameters fall within standard ranges (block S54). If set parametersfall outside these ranges, an error readout is generated (block S56),the system runs a diagnostic (block S58), and the procedure suns again.However, if a determination is made that the set parameters fall withinthe desired ranges, then the deconvolution algorithm is run (block S60).

At this juncture, a determination is required regarding whether the testcompound has an affect on the action potential. If the test compound hassubstantially no effect on the action-potential, then the cell typeand/or the medium conditions is/arc changed, as at block S64, and theprocedure is repealed (i.e., return to start). If the compound has anaffect on the action potential, a data report is output, as at S66, andthe procedure is repeated through block S52 to test a subsequent oranother compound.

It is important to recognize that concurrent with setting thephysiological state parameters, as at block S52, the system algorithm iscapable of running a biological subroutine, as at S68. Here, thissubroutine is generally concerned with cell integrity, where a systemcheck, as at S70, for example, can be performed on whether the cells arestill viable, since they must be exposed to correct osmolarity andtemperature. If the system check is negative, an error readout isoutput, as at S72, a diagnostic is run, as at S74, and the procedurereturns to start.

However, if the system check is affirmative, a feedback loop is set-up,as at block S76.

Detection and Determination of Unknown Agents. Application of the systemalgorithm Manipulation algorithm) to the function of potential drugs andother agents, can result in determination of which cellular processesare affected by the agent. In a still more particular embodiment of themethod of the invention, the deconvolution leads to information onpathways or functional categories affected in the cell.

Genomics Analysis. In a yet still more particular embodiment of thesystem, said accompanying software comprises instructions formanipulating one or more system parameters to alter one or moreconditions of a given experiment, for interpreting the outcome of suchmanipulations, or for both.

One embodiment of the invention is a system for high throughput analysiscomprising: a solid state microelectrode with an intervening layercomprising a surface modifying monolayer, which functions as a highimpedance seal; a cell culture which exhibits a measurable actionpotential; and means for biological analysis, that is, deconvolution, ofthe action potential of the cell culture. The cell culture can betransfected with a gene, an isolated nucleic acid, a fragment of anucleic acid, or combination thereof. In a yet further embodiment of theapparatus of the invention, the gene, isolated nucleic acid or fragmentof a nucleic acid is from a human.

In still another embodiment of the apparatus of the invention, thealgorithm comprises a feedback loop. The deconvolution and systemalgorithms are preferably stored on storage media, e.g., magnetic media(magnetic disk or tape) or optical media (CD-ROM).

5.8. Other Preferred Embodiments of the Invention

Another embodiment of the invention regards a kit for detecting an agentcomprising a sensor comprising a cell culture comprising at least onecell which exhibits a measurable action potential, a solid statemicroelectrode, an intervening layer comprising a surface modifyingmonolayer which functions as a high impedance seal; and software capableof biological based deconvolution of the action potential; and asampling device. In yet another embodiment of the kit of the inventionthe sampling device comprises an environmental sampling device,including, for example, an air pump.

Another embodiment of the invention regards a method of detecting anagent comprising: providing the kit, described above, and permitting anagent to interact with the sensor to detect the agent.

Another embodiment of the invention regards a kit for determining a genefunction comprising a system comprising a solid state microelectrode, anintervening layer comprising a surface modifying monolayer whichfunctions as a high impedance seal; software capable of biologicalanalysis of an action potential; and an algorithm capable of elucidatinga cellular pathway or receptor of interest to determine gene function;and a cell culture capable of hosting a transfection which exhibits ameasurable action potential; at least one gene for transfection; and atransfection facilitator. In yet another embodiment of the kit of theinvention, the transfection facilitator is an adenovirus or lipofectin.

Another embodiment of the invention is a computer readable mediumincluding instructions being executed by a computer, the instructionsinstructing the computer to execute a method of high throughputanalysis, the instructions comprising deconvolving an action potentialof a cell culture. In yet another embodiment of the computer readablemedium, the cell culture is adherent to a solid state microelectrode.

Another embodiment of the invention is a computer system for highthroughput analysts, the system comprising: a processor; a computerprogram controlling operation of the processor, the program includinginstructions for causing the process to effect deconvolution of anaction potential of a cell culture. In yet another embodiment of thecomputer system, the processor includes a network. In still anotherembodiment of the computer system the network includes an intranet, aninternet, an extranet, or a virtual private network. In a furtherembodiment of the computer system, the processor is linked by standardwire or standard wireless means to the network.

In a further embodiment, another, separate, computer readable medium isprovided which permits the parameters of the system to be changed and/ormanipulated such that experimental conditions can be varied. Inparticular, the instructions encoded into a preferred computer readablemedium are capable of customizing the system to provide desired outcomeson exposure of the system to one or more lest substances. Such a “systemsoftware” may make of other programs, including deconvolution software.It is important to note that while the present invention's deconvolutionsoftware preferably excludes spectral analysis, more specifically, aFourier transformation, the present invention's system software mayutilize such spectral analysis or Fourier transformation.

To further illustrate the present invention, the following examples areprovided for consideration by the reader.

6. EXAMPLES 6.1. Use of Cardiac Myocytes and Spinal Cord Neurons

Templates for accurate spatial placement of a neuronal cell network isprovided by the use of SAMs which permit application of a wide spectrumof circuit and fabrication technology to the detection of signals. Coldmicroelectrodes are used to measure signals from both cardiac myocytes(FIG. 1). Recordings are made at 37° C. The planar microelectrode arrayis platinized and coated with the cell permissive artificial substrate.Panel a illustrates a single action potential (AP) from a spontaneouslyfiring monolayer. Panel b illustrates a recording from the samemicroelectrode site, but over a longer time spun, thereby demonstratingpulsatility of the extracellular signal. Signals from spinal cordneurons are illustrated in (FIG. 2). The recordings demonstrate cellfiring initiated by depolarizing stimulus. Metal microelectrodes areused as substratum for neuronal cells in culture (FIG. 3). The neuronsare grown on a modified Si₃N₄ coated microelectrode, and signals arerecorded from Au microelectrodes in serum-free media. Thus cells arecultured in a defined media on a Si₃N₄ surface, the signals arerecorded, processed and displayed by the electronic interface. Thecell's activity on a microelectrode is attenuated by the introduction ofa toxin to the in vitro environment which is experimentalproof-of-concept for a neurotoxicity assay (Jung, D. R.; Cutlino, D. S.;Pancrazio. J. J.; P. Manos, P.; Custer, T.; Salhanoori, R. S.; Aloi, L.E.; Coulombe, M G.; Czarnaski, M. A.; Borkholdcr, D. A.; Kovacs, G. T.A.; Stenger, D. A.; Hickman, J. J. (1998), incorporated herein byreference, in its entirety. Cell-based sensor microelectrode arraycharacterized by imaging x-ray photoelectron spectroscopy, scanningelectron microscopy, impedance measurements, and extracellularrecordings. J. Vac. Sci. Technol. A, 16(3), May/June. 1183-88.).

The difference in signal-to-noise between FIGS. 1 and 2 is due to thefact that the monolayer formed by the larger cardiac cells establishes alarge seal resistance to the surrounding media by simply setting up amechanical barrier to ion transport while the single neurons in thespinal cord culture are much smaller and isolated and the ions can morefreely diffuse to the electrode surface. However, the resultsdemonstrate that the signals produced by the mammalian cells are strongenough to be readily detected by the microelectrodes. As the sealresistance is increased the signal-to-noise approaches that achievedwith glass micropipettes thereby allowing analysis of the AP waveformsfrom chip-based recordings.

6.2. Contribution of Ion Channels to Action Potentials

Another aspect of (his invention is the correlation of the shape of theaction potential with the pathways stimulated by biological responsemodifiers. Neurons exhibit a modified action potential when acted uponby different pathogens and toxins that are then recorded by anelectronic pickup. The NG108-15 (neuroblastoma×glioma) cell line, thatexhibits stable electrical activity upon chemical stimulation is studiedto show the effect of a wide variety of toxins on the action potential.The culture methods used and detailed electrophysiologicalcharacterization of this cell line are reported in Ma et al., 1998. Amodel system of an NG108-15 cell is illustrated in FIG. 4, whichindicates the relationship of toxins from various “functionalcategories” to various pathways compiled from the literature ((Becerril,B., Marangoni, S., Possani, L. D. (1997). Toxins and genes isolated fromscorpions of the genus Tityus. Toxicon 35, 821-35.; Brazil, O. V. andFontana, M. D. (1993). Toxins as tools in the study of sodium channeldistribution in the muscle fibre membrane. Toxicon 31: 1085-98.;Cantiello. H. F. (1995). Role of the actin cytoskeleton on epithelialNa⁺ channel regulation. Kidney Int. 48:970-84.; Cassola, A. C. andAfeche, S. C. (1996). Use of neurotoxins to study Ca²⁺ channelfunctions, Braz. J. Med. Biol. Res. 29: 1759-63.; Catterall W A, TrainerV, Baden D G. Related Articles Molecular properties of the sodiumchannel: a receptor for multiple neurotoxins. Bull Soc Pathol Exot.1992; 85(5 Pt 2):481-5.; Childers, S. R. and Deadwyler, S. A. (1996).Role of cyclic AMP in the actions of cannabinoid receptors. Biochem.Pharmacol. 52: 819-27.; Cowan, F. M., Shih, T. M., Lenz, D. E., Madsen,J. M., Broomfield, C. A. (1996). Hypothesis for synergistic toxicity oforganophosphorus poisoning-induced cholinergic crisis and anaphylactoidreactions. J. Appl. Toxicol. 16: 25-33.; Dryer, S. E. (1994).Na⁽⁺⁾-activated K⁺ channels: a new family of large conductance ionchannels. Trends Neurosci. 17: 155-60.; Faden, A. I. (1996). Neurotoxicversus neuroprotective actions of endogenous opioid peptides:implications for treatment of CNS injury. Nida Res. Monogr. 163:318-30.; Fields T A, Casey P J. Related Articles Signalling functionsand biochemical properties of pertussis toxin-resistant G-proteins.Biochem J. 1997 Feb. 1; 321 (Pi 3):561-71; Fozzard, H. A. and Lipkind,G. (1996). The guanidinium toxin binding site on the sodium channel.Jpn. Heart J. 37: 683-92.; Harvey. A. L. (1990). Presynaptic effects oftoxins. Int. Rev. Neurobiol. 32: 201-39.; Hille. B. (1994). Modulationof ion-channel function by G-protein-coupled receptors. Trends Neurosci.17: 923-42.; Holstege, C. P. Kirk. M., and Sidell, F. R. (1997).Chemical warfare. Nerve agent poisoning. Crit. Care Clin. 13: 923-42.;Janiszewski, L. (1990). The action of toxins on the voltage-gated sodiumchannel. Pol. J. Pharm. 42: 581-8.; Kallen, R. G., Cohen, S. A. andBarchi, R. L. (1993). Structure, function and expression ofvoltage-dependent sodium channels. Mol. Neurobiol. 7: 383-428.; Lewis,R. J. and Holmes, M. J. (1993). Origin and transfer of toxins involvedin ciguatera. Comp. Biochem. Physiol. C. 106: 615-28.; Mori, Y. G.Mikala, G. Varadi, G., Kobayashi, T., Kosh, S. Wakamori, M. Schwartz, A.(1996). Molecular pharmacology of voltage-dependent calcium channels.Jpn. J. Pharmacol. 72: 83-109.; Narahashi, T., Frey, J. M., Ginsburg, K.S., and Roy, M. L. (1992). Sodium and GABA-activated channels as thetargets of pyrethroids and cyclodienes. Toxicol. Lett. Narahashi, T.,Roy, M. L., and Ginsburg, K. S. (1994). Recent advances in the study ofmechanism of action of marine neurotoxins. Neurotoxicology 15: 545-54.;Nestler, E. J. Alreja, M., and Aghajanian, G. K. (1994). Molecular andcellular mechanisms of opiate action: studies in the rat locuscoeruleus. Brain Res. Bull. 35: 521-8.; Norton, R. S. (1991). Structureand structure-function relationships of sea anemone proteins thatinteract with the sodium channel. Toxicon 29: 1051-84.; Pearson, H. A.,Campbell, V., Berrow, N., Menon, J. A. and Dolphin, A. C. (1994).Modulation of voltage-dependent calcium channels in cultured neurons.Ann. N.Y. Acad. Sci. 747: 325-35.; Pfister, C., Bennett, N., Bruckert,F. Catty, P. Clerc, A., Pages, F. and Deterre, P. (1993). Interactionsof a G-protein with its effector: transducin and cGMP phosphodiesterasein retinal rods. Cell Signal 5: 235-41.; Piek, T. (1990). Neurotoxinsfrom venoms of the Hymenoptera—twenty-five years of research inAmsterdam. Comp. Biochem. Physiol. C. 96: 223-33.; Rizzo, M. A., Koesis,J. D., and Waxman, S. G. (1996). Mechanisms of paresthesiae,dysesthesiae, and hyperesthesiae: role of Na⁺ channel heterogeneity.Eur. Neurol. 36: 3-12.; Rowan, E. G., and Harvey, A. L. (1996). Toxinsaffecting K⁺ Braz. J. Med. Biol. Res. 29: 1765-80.; Savolainen K M,Hirvonen M R. Second messengers in cholinergic-induced convulsions andneuronal injury. Toxicol Lett. 1992 December; 64-65 Spec No: 437-45.;Schantz, E. J. and Johnson, E. A. (1992). Properties and use ofbotulinum toxin and other microbial neurotoxins in medicine. Microbiol.Rev. 56: 80-99.; Smith, B. A. (1990). Strychnine poisoning (publishederratum appears in J. Emerg. Med. 1991, November-December; 9(6): 555).J. Emerg. Med. 8:321-5.; Solberg, Y. and Belkin, M. (1997). The role ofexcitoloxicity in organophosphorous nerve agents enetral poisoning.Trends Pharmacol. Sci. 18: 183-5.; Swift, A. E. and Smith, T. R. (1993).Ciguatera. J. Toxicol. Clin. Toxicol. 31: 1-29.; Uchitel, O. D. (1997).Toxins affecting calcium channels in neurons. Toxicon 35: 1161-91.; Van,H. H., Busker, R. W., Melchers, B. P., and Bruijnzeel. (1996).Pharmacological effects of oximes: how relevant are they? Arch. Toxicol.70: 779-86.; Wu. M. (1997). Enhancement of immunotoxin activity usingchemical and biological reagents. Br. J. Cancer 75: 1347-55.; Yoshida,S. (1994). Tetradotoxin-resistant sodium channels. Cell Mol. Neurobiol.14: 227-44)). In FIG. 4, bold face indicate toxins that have beentested. These data are also represented in Table 1. The significance ofthese results is that not only obvious neuronal toxins such astetrodotoxin (TTX) affected the AP but also many others not so obvious,such as ricin. Out of 14 functional categories (Table 1), six affect theAP, including agents that modulate cellular processes, signalingpathways, transcription, is indicated. All of the listed toxins exceptVX lead to a cessation of the action potential within 180 seconds.However, differences are noted on the way the action potential ceaseswhich we have used to determine which pathway and ultimately which ionchannel is primarily affected by a particular toxin. This is illustratedin FIG. 5 which shows the AP after the administration of paraoxin, atransport and binding protein; ricin a protein synthesis inhibitor; andcyanide an energy metabolism inhibitor. FIG. 5 shows (top) the loss ofsignal upon administration of 3 mM paraoxon that shows a slowdown ofsignal or change in phase, the depolarization at the membrane, and onsetof blockage in <60 s, which wits not reversible after toxin washout. Thestimulated action potential using intercellular recording (middle) showsthe dramatic changes in action potential shape for the duration,amplitude (AMP), and after hyperpolarization potential (AHP). Thus,identification of the cellular pathway by detailed interpretation of therecorded signal forms the basis of diagnostic concept of the presentinvention for identifying electrophysiological data and relating them to“cellular function” categories using our deconvolution algorithms.

An action potential is altered or interrupted in different ways bydifferent toxins corresponding to interruption of ion channels in apathway specific manner. Comparison of APs following the administrationof different biological response modifiers or agents on cells leads tothe identification of pathways for further development of analysisalgorithms. The pathway determination involves parameters such as theshape change in the AP, time to cessation of activity, frequency andamplitude changes, and other factors.

The analysis of action potential signals is a sensitive indicator of thebiochemical pathway or functional category involved. The logic is thatthe effects of some classes of toxins is to change ion conductanceparameters (e.g. magnitudes or lime constants) with resulting changes inthe extracellularly recorded waveshapes and spike rates. Signals arerecorded which are either reduced amplitude copies of the membranepotential (when the seal resistance is high) or of the derivative of themembrane potential (when the seal resistance is low). To illustrate animplementation of the concept simulations of the Hodgkin-Huxley modelare created in which the time constant of the sodium channel is doubled,and in which the potassium channel time constant is increased by afactor of five. The simulated extracellular somatic waveforms are eitherthe membrane potentials (FIG. 6-a) or their derivatives (FIG. 6-b). InFIG. 6 the largest peak is from a simulation in which the potassiumchannel time constant was lengthened by a factor of five—note the longeraferpotential. The smallest of the peaks results from increasing thesodium time constant by a factor of two. The remaining peak is thenormal ‘textbook’ Hodgkin-Huxley simulation. The slowing of the sodiumchannel decreases the signal amplitude, widens its main peak, but doesnot affect the afterpotential. The slowing of the potassium channelslightly increases the fast sodium peak, lengthens that peak but not itsderivative, significantly lengthens the afterpotential, and greatlyreduces the spike rate under constant stimulation (the latter notshown). These results clearly indicate that the waveform shape is verysensitive to even small variations in the conductance. Thesesimulations, combined with the toxin data indicate the direct connectionbetween the receptor effecting the pathways, the ion channels and the APshape. These data are the beginnings of a library that will be formedfrom testing known compounds or drugs in our system and cataloging thechanges in AP shape. The deconvolution algorithm will utilize theselibraries to identify the functional categories and pathways affected byunknown compounds or genes.

6.3. Cell Types Useful in the Invention

Any electrically active cell can be used as a diagnostic element. One ofthese could be a cell line. The NG-108-15 cell line, used isderived froma glioma×neuroblastoma hybridand has been shown to provide reproducibleresults. Most of the data has teen collected with this cell type andthey have been shown to live two to three months in our defined culturesystem. The lifespan of the primary CNS cells in our defined orreproducible cell culture system is about a month. Primary cells,however, have the advantage of more closely approximate in vivo systemsthan tumor-derived cell lines. A solution that combines the favorableaspects of these two options is the utilization of clonal lines derivedfrom CNS stem cells. Companies, such as NeuralStem and BioWhitaker, havedeveloped stable cell lines of CNS neurons from stein cells and havetransformed primary neuronal cells into cell lines.

The electrical characteristics of all of these cells can be monitored onthe microelectrode arrays. In addition to the remarkable ability tomaintain the stability of the intrinsic neuronal character through manycell divisions, CNS stem cells are remarkably plastic; that is, a singleextracellular factor can shift the fate specifications of the cells intolargely one cell type or another. Thus, having a stable long-lived cellphenotype in combination with novel and advanced surface chemistry forspecific placement of cells on microelectrode arrays for signaltransduction forms key components of the assay system. Since eachindividual cell becomes a unique assay element and as the cells arelocalized on individual microelectrodes on a chip, statistics areperformed on a reproducible population of cells in response to thecompound or protein being examined.

6.4. Patch Clamp and Solid State Microelectrode-Based Electrophysiologyto Enable

Cellular Category Elucidation

Cellular function in relation to changes in the neuronal actionpotential after biochemical introduction is determined by usingbiochemical “triggers” that each activate a distinct pathway in thefunctional categories, described above. The system is established byreproducible changes in the shape of the action potential that varieswith some of the genetic categories described in 6.4.1 Further supportis provided in FIGS. 4-7. Moreover, a subset of the major categoriesbased on sequence homologies in the model system, can determine two“triggers” for each pathway, monitor the AP in the presence of thesecompounds, and demonstrate unique AP signatures for the pathways. Asimple system such as bacterium can define a simple genetic basis forthe evaluation.

TABLE 1 Assessment of Bio-Agents on NG-108-15 Cells Onset CompoundEffect Concentration Time I. Transport/Binding Proteins tetrodotoxin*Na⁺ channel 100 nM <60 sec. brevetoxin* Na⁺ channel 10 μM <60 sec.apamin* K_(Ca) “SK”-type channel 10 μM <60 sec. quinine* non-specific K⁺channel 2 mM <60 sec. Charybdotoxin* K_(ca) “BK”-type channel 10 μM <60sec. VX reversible attenuation 10 μM <240 sec . Paraoxon irreversible 3mM <60 sec. depolarization DFP irreversible 250 μM <60 sec.depolarization II. Cellular Processes verapamil* L-type Ca²⁺ channel 0.5μM <60 sec. nifedipine* L-type Ca²⁺ channel 0.5 μM <60 sec. ω-conotoxin*N-type Ca²⁺ channel 10 μM <60 sec. amiloride* L-type Ca²⁺ channel 10 μM<60 sec. carbachol* muscarinic receptor 10 μM <60 sec. III. CellEnvelope/Membrane Palytoxin irreversible 1 μM <60 sec. depolarizationIV. Regulatory Function ouabain* Na⁺—Ca²⁺ pump 2 mM <60 sec. inhibitionV. Translation Ricin irreversible 10 μM <180 sec.  depolarization VI.Energy Metabolism Cyanide reversible depolarization 100 μM <60 sec.*indicates compounds which inhibit repetitive firing.

6.4.1. Cellular Categories from Genetic Data

The genetic categories based on sequence homologies defined forbacteria, are used initially to provide subsets of function foranalysis. Of the 14 genetic categories defined by Riley et al. (Riley,M. (1993) Functions of gene products of Escherichia coli. Microbiol.Rev. 57, 862-952.), we use as examples groups most relevant to cellregulation, although all 14 categories can be investigated with themethods of the invention. Six broad categories have been selected: (a)Energy Metabolism, (b) Amino acid biosynthesis, (c) Cellular processes(d) Fatty acid, phospholipid, and steroid Metabolism, (c)Transcriptional regulation and (f) Transport and binding proteins. Theneurons or cardiac myocytes are monitored for changes in the AP by glassmicroelectrodes in an on-cell extracellular recording mode or bymicroelectrode arrays. Signals corresponding to changes in the membranepotential are deconvolved using the methods described above. Twocellular preparations are particularly useful in the invention. First,cultured NG-108-15 embryonic hippocampal neurons are used. Analysis ofthese data identify characteristics of each category. A second set ofdata are collected for cells localized on the microelectrode array. Thisdesign permits determination of action potential shape changes that arecharacteristic of these six broad classes of compounds and how cells onthe microelectrode array respond to stimuli in comparison to the wellestablished cultured cell model.

6.4.2. Experimental Design and Detailed Compound Selection Rationale

Studies are performed with each test compound or “trigger” to determinean effective concentration. Effective concentrations cause aphysiological response without causing cell death, within 12 hours ofthe initial exposure. The studies are conducted 7 to 10 days after cellplating. Unless otherwise noted, cells in the Examples are cultured andmaintained in serum free media containing 10 mM glucose as describedpreviously (Schaffner, A., Barker, J. L., Stenger, D. A., and Hickman,J. (1995). Investigation of the factors necessary for growth ofhippocampal neurons in a defined system. J. Neurosci. Methods, 62,111-119), although other media formulations are known to those skilledin the art.

Extracellular clamp electrophysiology is used to monitor how the actionpotential changes when the hippocampal neurons are exposed to differentbiochemical “triggers”. These changes are used to map certain landmarkevents that occur intracellularly that are also related to theelectrical activity of the individual ion channels whose collectiveactivity comprise the action potential. This information is used togenerate first stage algorithms to deconvolute the shape of the actionpotential and relate it to the pathways or categories (see below for APwaveform analysis). Specific examples of trigger agents and regulatorycategories follow. One skilled in the art will be able to use otheragents not listed here based on these examples.

6.4.2.1. Energy Metabolism-2-deoxy-D-glucose (2-DG)

A number of studies using adult hippocampal AC1 neurons have shown thattemporary block of glycolysis by 2-deoxy-D-glucose (2-DG) reversiblysuppresses synaptic transmission in the CA1 region of hippocampalslices. When the neurons recover, a sustained potentiation of fieldexcitatory postsynaptic potentials (EPSPs) is observed. Thus, 2-DG is amolecule capable both of directly affecting the action potential and ofinducing changes in gene expression and protein synthesis based on itsmetabolic action to block glycolysis.

The media in the apparatus of the invention is supplemented 24 hoursbefore the start of the signal collection by adding 5 mM ketone bodiessupplied as 2:1 ratio of β-hydroxybutyrate: acetoacetate as an energysource. Initial 2-DG is added to provide a 10 mM concentration.Additional inhibitors of energy metabolism acting at different sites canequally be used, including malonate, a compound that blocks energyproduction in the tricarboxylic acid cycle through NADH generation and2,4-dinitrophenol, an uncoupler of oxidative phosphorylation.

Additional triggers are glucose and fructose. Glucose is a broadfunction energy metabolite but yields effects on the action potentialwith concentration changes. Comparison of glucose effect with fructoseeffect on the action potential has implications for the pathways and forparticular disease states including diabetes and hypoglycemia.

6.4.2.2. Amino Acid Biosynthesis—Amino-oxyacetate

Amino-oxyacetate is a well described inhibitor of transaminase activity,notably those reactions transaminating glutamate. Although it ispossible to synthesize both glutamate and GABA without transaminaseactivity, significant alterations in nitrogen metabolism are produced bythis inhibitor. Such alterations can lead to changes in gene expressionas a compensation to the effects of this compound.

Amino-oxyacetate is used at 5 mM with the system of invention.[15N]-glutamine labeled in the amine or amide N is included in smallaliquots of cells and the recovery of ¹⁵N in glutamate, aspartate, GABAor alanine is monitored with gas chromatography/mass spectrometry ofthese amino acids to verify alteration of transamine activity. Transferof ¹⁵N from the main N of glutamate to other amino acids is blocked byan inhibitor of transaminase. The effect of inhibition of proteinsynthesis on the ¹⁵N patterns is used to correlate the action potentialcharacteristics with the degree of change in these pathways.

6.4.2.3. Cellular Processes:—Cholecystokinin (CCK)

CCK is co-localized with the inhibitory neurotransmitter GABA ininterneurons of the hippocampus. CCK receptors are found in abundance inthe hippocampus and are known to antagonize the excitatory effects ofopiates. A recent report found that the sulfated octapeptide CCK-8Sincreased action potential frequency or generated inward currents in themajority of hippocampal interneurons (Miller K K. Hoffer A, Svoboda K R,Lupica C. R. Related Articles Cholecystokinin increases GABA release byinhibiting a resting K+ conductance in hippocampal interneurons. JNeurosci. 1997 Jul. 1; 17(13):4994-5003). As CCK is known to have acomplex functions in the brain including a role in satiety and possiblya detrimental role in ischemic damage, this compound provides a classicreceptor mediated peptide type hormone with direct and immediate effectson the action potential.

CCK-8S peptide is used in the invention at concentrations known toaffect the actions of opiates. 100 nM. The effects of a proteinphosphatase inhibitor, GTP γS (300 nM) and a protein kinase inhibitor,okadaic acid (100 nM) are investigated to further clarify the CCK-8response. Moreover, other peptide hormones acting via receptors on thehippocampal membrane such as neuropeptide Y, vasoactive intestinalpeptide, and transforming growth factor-β are used in the system of theinvention.

6.4.2.4. Fatty Acid and Phospholipid Metabolism/Cholesterol Synthesis,HMG CoA Reductase Inhibitor: Lovastatin

Neuronal cells synthesize cholesterol dc novo using acetyl CoA (Edmondet al. 1991). This pathway is highly regulated and the most importantcontrol step is at the enzyme HMGCoA reductase. De novo lipogenesis andcholesterol synthesis are particularly important in late fetal life andthe early post natal period. Messenger RNA for key enzymes in thecholesterol synthesis pathway including HMG-CoA reductase, farnesylpyrophosphate synthase, and cholesterol 7 α-hydroxylase increase duringthis period. In addition, lipophilic HMG CoA reductase inhibitors suchas lovastatin affect the nervous system in the areas of sleep andcognitive function. As cholesterol is an important component of the cellmembrane alterations in the shape of the action potential may be relatedto changes in the capacity to synthesize cholesterol.

Lovastatin is included in the tissue culture medium of the invention at10 μM, a concentration that blocks HMG CoA reductase in cultured cells.To further clarify efficacy of this compound in blocking cholesterolsynthesis a stable isotope method is used to quantify the actions oflovastatin on cholesterol synthesis. Moreover, other cholesterolsynthesis inhibitors such as 25-OH-cholesterol and other modulators oflipid metabolism, including the carnitine palmitoyl transferaseinhibitor TGDA and the anti-tuberculosis agent isoniazid, are used inthe system of the invention.

6.4.2.5. Transcriptional Regulation—Corticosteroids

The modulation of neuron excitability by corticosteroids especially inhippocampal subfield CA1 is well documented (Okuhara D Y, Beck S G.Related Articles Corticosteroids alter 5-hydroxytryptamine 1Areceptor-effector pathway in hippocampal subfield CA3 pyramidal cells. JPharmacol Exp Ther. 1998 March; 284(3): 1227-33). The hippocampuscontains the highest density of mineralocorticoid and glucocorticoidreceptors in the central nervous system. Corticosteroids regulate geneexpression through the activation of nuclear mineralocorticoid andglucocorticoid receptors.

Corticosterone is used in the invention for testing ion balance asreflected in the electrical activity of the cells. Moreover, othersteroids, including aldosterone, dexamethasone and RU38486 are used inthe system of invention.

6.4.2.6. Transport and Binding Proteins—Cholinergic Agonists, Carbachol

Cholinergic input to the hippocampus from the medial septum plays a keyrole in modulating hippocampal activity and functions, including thetarhythm and spatial learning. Recently it has been found that thecholinergic agonist carbachol caused several reversible changes in theaction potential recorded from CA1 pyramidal cells in hippocampal slices(Figenschou A, Hu G Y, Storm J F. Related Articles Cholinergicmodulation of the action potential in rat hippocampal neurons. Eur JNeurosci. 1996 January; 8(1):211-9).

Carbachol, the cholinergic agonist, is used in the invention to probethe cholinergic receptor at 2 μM. Moreover, other cholinergicantagonists such as pilocarpin or atropine, and agonists such asacetylcholine or muscarine, and partial agonists such as pilocarpine areevaluated.

Ion channel blockers are used to modulate the action potential. TEA(e.g., tetraethylammonium bromide) is very specific for the potassiumchannel. Similarly, TTX (tetrodotoxin) is specific for the sodiumchannel. TTX can block the spontaneous electrical activity of neuronsand is a reversible blocker. The TTX-induced blockade of the electricalactivity can result in an altered gene expression pattern in thedeveloping neuron. Agonists of L-type voltage sensitive calcium channelsare used to block effects of TTX.

6.4.3. Elements of the Biological Component

6.4.3.1. Creatine a Defined System and Neuronal Characterization

Hippocampal neurons derived from embryonic day 18 CNS stem cell neurons,other neuronal cells, cardiac myocytes and other cells with a measurablemembrane potential change are useful in the invention. NG-108-15 cellshippocampal neurons have been extensively used as models in variousneuronal culture systems. They express the necessary neurotransmitterreceptors and ion channels critical for the purpose of this invention.Hippocampal neurons are terminally differentiated, non-dividing cells.CNS stem cells can be differentiated into a neuronal-like phenotypewhich will express several of the desired neurotransmitter receptors andion channels.

The cells are cultured on homogeneous SAM surfaces. DETA has beenreported to be the best artificial surface for short term (<1 month)hippocampal culture. (Schaffner. A., Barker, J. L., Stenger, D. A., andHickman, J. (1995). Investigation of the factors necessary for growth ofhippocampal neurons in a defined system. J. Neurosci. Methods, 62,111-119.) B27-supplemented neurobasal medium is used for neuronalgrowth, and the cells are incubated in 5% carbon dioxide in air (v/v) tocreate a defined scrum-free system (Brewer G J, Torricelli J R, Evege EK, Price P J. Related Articles Optimized survival of hippocampal neuronsin B27-supplemented Neurobasal, a new scrum-free medium combination. J.Neurosci. Res. 1993 Aug. 1; 35(5):567-76). This serum-free systemselects against glial cells. Medium is changed twice a week to insurehealthy cultures.

The neurons in the apparatus of the invention are characterizedmorphologically, immunocytochemically, and by extracellularelectrophysiological recordings to evaluate changes in the membranepotential. A poly-D-lysine (PL) standard is run for each analysis toevaluate the general health of the original cell suspension.

6.4.3.2. Characterization of Neuronal Morphology

The neurons in culture are characterized according to several criteriadescribed below. Cell survival and morphological characterization isaccomplished by photographing the culture dishes at 24 hours afterplating. Cell survival is assessed by comparing the number of cellssurviving at a given point relative to the initial number of cells. Thefollowing quantitative values are also measured.

6.4.3.3 Cell-to-Substrate Surface Contact Area

Contact area of well separated cells is measured by marking the cellboundaries on the image and calculating the enclosed areas. A moreadhesive substratum results in flatter, more adherent cells and thus alarger area in contact with the substratum.

6.4.3.4. Extent of Aggregation

As a measure of migration and cell-cell adhesion, aggregation isevaluated by measuring the total surface occupied by clusters organglion-like structures where contact inhibition has been lost.

To analyze these data the an imaging program is used which allows (1)drawing boundaries around cell somas or aggregates to calculate area, ordrawing a boundary just outside the cell soma to calculate intersectionsand determine the number of primary neurites, (2) drawing the frequencyhistograms, and (3) performing a chi-square analysis to determinewhether distributions differ with different protocols. An analysis ofvariance is performed to determine if there are treatment differences aswell as plate differences. This permits determination the number ofreplicates necessary to generate statistically meaningful results.

6.4.3.5. Immunocytochemistry: Identification of glia in Culture

Cells are characterized immunocytochemically for neuron-specificantigens (neurofilament neuron-specific enolase, Tujl), anastrocyte-specific antigen (glial fibrillary acidic protein), and anoligodendrocyte-specific antigen (galactocerebroside).

6.4.3.6. Neurotransmitter Expression

Neurons are characterized with respect to neurotransmitter expressionwith commercially available antisera to the neurotransmitters GABA andglutamate. The immunocytochemistry experiments is performed at days 7and 10 after plating.

6.4.4. Differentiating Gene Expression

For detecting changes in gene expression, the experimental protocolallows facile detection of differences in action potential resultingfrom changes in gene expression. Thus, five test conditions areroutinely compared for each compound:

(a) Control

(b) pre-incubated for 3 hrs with cyclohexamide, a well described proteinsynthesis inhibitor;

(c) +test compound alone;

(d) pre-incubated for 3 hours+test compound; and

(c) pre-incubated for 3 hours+cyclohexamide+test compound

This strategy distinguishes those responses to the test compound thatare immediate and not likely to be related to changes in proteinsynthesis (by comparison of 1 and 3) versus those responses that requiteprotein synthesis (by comparison of 4 and 5), with consideration of anyeffect of cyclohexamide.

6.4.5. Glass Microelectrode Recording from Neurons

Extracellular recordings that mimic the conditions used for on-chiprecording are applied to neuronal cells. Changes in the membranepotential in a “maxipatch” mode which is an on-cell patch where agigaohm sea) is formed to keep the noise low and enough of the cellmembrane is contacted to make the ion channels representative areperformed. The recording solution is the same as the extracellularmedium in order that the membrane experience as normal an environment aspossible. The potential in the pipette, which approximates the membranepotential, is recorded (current clamp mode, with zero current). (Thecircuit formed by the membrane, amplifier, and seal impedances ismodeled to correct for differences between the potential recorded by thepipette and the presumed true membrane potential). The neurons areinduced to fire by the addition of 40 mM KCl in lieu of electricaldepolarization of the membrane. Mg²⁺ is added to the media beforerecording to inhibit synaptic transmission. The electrophysiologicalproperties of hippocampal neurons at 7 days after plating and 10 daysafter plating are compared. Other time points are examined as necessary.

6.4.6. Deconvolution Analysis of Action Potential Peak Shapes

The general methods for AP detection and sorting have recently beenreviewed. (Wheeler, B. C. (1999). “Real Time Techniques for AutomaticDiscrimination of Single Units”, book chapter, in press, to Methods forNeural Ensemble Recordings, M. Nicolelis (editor), CRC Press.) Analysis,i.e., deconvolution, of action potentials is based on a complete suiteof algorithms, including sorting by amplitude, template matching andprincipal components, as well as automated cluster cutting to aid indetermining the number of distinct APs in a recording and theirwaveshapes; also included are algorithms for extracting various spiketrain features (e.g. spike rate, burst rate and burst duration);stimulus driven measures (e.g. peristimulus time histograms,input/output functions); and inter-neuron correlation measures (e.g.cross-correlograms, mutual information).

The signal processing is performed by recording and analyzing actionpotentials before, during and after treatment with the appropriatetoxins at prescribed concentrations. In the first step the changes inwaveshape—e.g. measures of width, height, spike rate—are described asfunctions of the “trigger,” concentration, and the cellular category onwhich the toxin acts. A model is applied to estimate the seal resistancefrom the recorded waveform data to assist in the interpretation.Secondly, a Hodgkin-Huxley type model is applied using conductancemechanisms and data in the literature for hippocampal pyramidal neurons.The parameters of the model are optimized to fit the acquired waveformsand waveshape measures. In the third stage the uniqueness of the changesas indicators of altered membrane physiology is evaluated; for instancein the example simulations, primary peak width is a good indicator ofchanged potassium dynamics when the seal resistance is high, but not ifit is low (conversely, afterpotential duration should be more usefulwhen the seal resistance is low). In all cases, statistical evaluationis performed, estimating the sensitivity of the measure to changes inmembrane physiology with reference to the irremovable variation—bothwith instrumentation noise and variation from cell to cell.

6.4.6.1. Effect of Potassium Channel Modulators

Solid state microelectrode arrays are prepared having 32 goldmicroelectrodes and leads accessed by means of 14 μm diameter viasthrough a 1 μm thick silicon nitride lop layer on a silicon base. Thearrays are chemically cleaned prior to electroplating and again prior tosubstrate modification with silane monolayers. First, the arrays arerinsed with de-ionized water and then with high purity liquidchromatography (HPLC) grade acetone. Then, each array is soaked inhexane for five minutes and then rinsed three times in acetone.Following these latter steps, the arrays are immersed in concentratedsulfuric acid: 30% (v/v) hydrogen peroxide (4:1 v/v) for two minutes andthen rinsed five limes with HPLC grade water and three times with HPLCgrade acetone. Microelectrodes are then electroplated with platinumblack by standard procedures. The silane self-assembled monolayer isapplied by reaction of the arrays for 15 minutes with 1% (v/v) of(aminoethylaminomethyl)-phenethyltrimethoxy silane in 94% (v/v) 1 mMacetic acid in anhydrous methanol and 5% water. A solution is preparedconsisting of 1% (w/v) each of antibody to N-Cadherin, and, optionally,antibody to R-Cadherin, antibody to adult and embryonic pan-N-CAM, andantibody to neurite cell adhesion molecule L1 in 50 mM phosphate buffer,pH 7. This solution containing antibodies is incubated with the arrayfor one hour in the absence or presence of 1 mM carbodiimide. The arrayis then rinsed once with HPLC grade water and once with Neurobasalmedium. Hippocampal neurons are obtained by enzymatic digestion usingpapain (2 units/ml) from embryonic day 18-19 rat pups. Neurons areplated on substrates at a density of 1 to 1.5×10⁴ cells/cm² and culturedin Neurobasal medium supplemented with 2% (v/v) B27, 0.5 mM glutamine,and 25 μM glutamate.

The impedance of each electrode is measured as described in Example6.5.5. Each microelectrode with a suitably high impedance is used forfurther measurements. The action potential elicited by 2 μM carbacol isevaluated and compared to the action potentials elicited by 2 μMcarbacol in the cells incubated in the presence of 1-10 μM apamin, 1-10μM charybdotoxin, or 1-10 mM TEA. The contribution of potassium channelsto the action potential is determined by the analysis methods describedin 6.4.6., in particular analysis of changes in waveshape followed byapplication of a Hodgkin-Huxley model. The potassium channel function isfurther analyzed in terms of the concentration and type of blocker usingthe system algorithm (manipulation algorithm).

6.4.6.2. Analysis of Cyclic-AMP Regulated Pathways by their Modulationby Cholera Toxin

Analysis of integrated or higher order pathways is accomplished bybuilding on the analysis of ion channels as in 6.4.6.1., and similaranalysis of sodium and calcium channels, in the higher order analysis(system analysis), several ion channels can be affected. For example,the effect of cholera toxin on the action potential is evaluated asfollows. Cholera toxin is known to have a primary effect on theG-protein of adenylate cyclase, resulting in persistent activation ofthe enzyme and production of cyclic AMP. Using the system described inExample 6.4.6.1., the effect of graded doses of cholera toxin on theaction potential of hippocampal neurons is evaluated. The changes in theaction potential reflect, in part, changes in ion flux in the sodium,potassium, and calcium channels.

6.4.6.3. Analysis of Unknown Agents by Their Effect on Action Potentials

A collection of agents of unknown function derived from hunter snails,and other animals, from dc novo synthesis, from medicinal plants, andelsewhere, is evaluated using the system described in Example 6.4.6.2.The effects of the agents on hippocampal action potentials are rapidlycompared to the effects induced by toxins with known mechanisms ofaction. Thus, an agent is described as apamin-like, or choleratoxin-like, etc., based on the pathways and ion channels that areaffected. Moreover, agents that affect multiple pathways can be analyzedin terms of the complex effects on the action potential.

6.4.6.4. Metabolic State as a Preexisting Condition for FurtherAnalysis: Cross-Effects of Experimental Drugs with Fluctuations inGlucose and Insulin

Metabolic states are important determinants of drug action and efficacy,yet are poorly modeled with current in vitro assays. Among the moresignificant metabolic states are those reflective of activity, such asexercise; eating or fasting; carbohydrate or fatty diet; and disease,such as diabetes. Physiological states corresponding to fasting andeating are modeled by reducing and increasing, respectively, glucoseconcentrations in the medium, concomitant with modulation in insulinconcentrations. Similarly, ketoacidosis, hypoglycemia, hyperosmolarity,lactic acidosis, and other metabolic states can be modeled by oneskilled in the art. The effects of normal and pathological states,including hypoglycemia and diabetes, on neuronal pathways aredetermined. Thereby, the rapid analysis of complex drug interactions atdifferent metabolic states is possible.

After anchorage of hippocampal neurons or cardiac myocytes on solidstate microelectrodes, the impedance is monitored to insure formation ofu high impedance seal. KG is added to stimulate action potentials.Likewise, neurotransmitters or a shift in pH can be used elicit actionpotentials. The resultant action potentials are deconvolved by analgorithm for analysis of the biological response of the system. Theculture medium bathing hippocampal neurons on the solid statemicroelectrode is then replaced, in a step-wise fashion, with Neurobasalmedium with modified glucose concentrations, supplemented with 2% (v/v)B27, 0.5 mM glutamine and 25 μM glutamate. The medium is prepared with25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.20 mM glucose. Moreover,the cells are exposed to graded doses of insulin. Then the actionpotentials are again analyzed at each glucose and insulin concentrationpair to elucidate the effects of glucose and insulin on ion channelfunction. KCl is used to initiate the action potentials. At a firstphase of system analysis, the effect of glucose-insulin pairs oncellular processes is determined by comparison to the actions of knowneffectors. Then drugs can be screened or further evaluated. For example,acarbose, an alpha-glucosidase inhibitor sometimes used for reactivehypoglycemia and which reduces postprandial blood glucose and theinsulin response is added to the hippocampal cultures. Similarly anentire combinatorial library of agents can be screened. Upon generationand recordation of action potentials a second phase analysis of theinvolvement of cellular processes is initiated. Thus the specificeffects of the complex combination of acarbose with different levels ofglucose and insulin are determined. Similarly, agents can be identifiedfrom a combinatorial library as effective in particular metabolicstates.

By similar methods, the effect of clinical and experimental agents fortreatment of diabetes are evaluated. The resulting information isparticularly valuable for patients with multiple diseases orcomorbidities who are subject to complex pharmaceutical interactions.For example, repaglinide is an agent that restores euglycemia by anaction on ATP-dependent potassium channels in the beta cells of thepancreas. Graded doses of repaglinide are added to the system of theinvention to rapidly analyze the effects of repaglinide on neuronalaction potentials under a variety of metabolic states, includinghypoglycemia, hyperglycemia, hypoinsulinemia, and hyperinsulinemia.Similarly one skilled in the art will understand that other agents canbe tested using the system of the invention.

Hippocampal neurons anchored to the microelectrode are evaluated forformation of a high impedance seal. Action potentials are initiated byaddition of KCl or carbacol, although other stimulators andneurotransmitters are equally useful. The resultant action potentialsare analyzed to determine the relative involvement of specific ionchannels. The culture medium bathing the neurons is varied so that theneurons are exposed to graded doses of glucose and insulin. In a firstphase of system analysis, the effect of metabolic state on cellularprocesses is evaluated. Then the cells are exposed, at eachglucose-insulin concentration pair, to repaglinide, to TGA, and to otherneuromodulators of known function. In a second phase of system analysis,the complex interactions of repaglinide, insulin, and glucose areresolved to provide improved dosing regimens for clinical use.Similarly, a panel of other agents useful in disease treatment,including troglitazone and sulfonylurea is evaluated to select agentsoptimally effective for each metabolic or disease state.

6.4.6.5. Modification of the Viscosity of the Interface Layer

The viscosity of the interface layer can advantageously be increased inseveral ways. A moderate or high viscosity interface layer between celland the silane layer on the microelectrode is associated with increasedimpedance of the seal.

In one method, a thickening agent is added to the bulk medium duringattachment of the cells and subsequently the bulk solution is rinsedaway with culture medium. Some of the agent is trapped between the celland the surface of the microelectrode array chip. Thus in preparationfor plating of hippocampal neurons, a culture medium containing between0.1 to 10% (w/v) hydroxyethyl starch, preferably 1%, is used until thecells have attached and the high impedance seal is established.

In another method, a viscosity enhancer is bound to the silane layer onthe microelectrode. Streptavidin is covalently linked to the aminogroups of the silane by standard cross-linking chemistry. Then themicroelectrode is exposed to biotin-conjugated antibody at a ratio ofabout 1:1 to about 1:0.01 (moles of antibody to moles of boundstreptavidin) optimally about 1:0.1. After the antibody is bound (30minutes at room temperature) to the streptavidin on the silane layer,the microelectrode array is exposed to a slight molar excess (over boundstreptavidin) of biotin-conjugated viscosity agent, bis(polyoxyethylenebis[biotin], which has a molecular weight of about 20,000 Daltons. Aftera 30 minute incubation, excess reagent is rinsed away with HPLC waterfollowed by a rinse with culture medium. Then cells (neuronal cells,NG-108 cells, or cardiac myocytes are equally effective) are seeded ontothe microelectrode array. After the formation of a high impedance sealis verified by measurement on impedance, the hybrid bio-electrode orapparatus of the invention is ready for use.

6.4.6.6. Microelectrode Array Diagnostic Test: Cellular and ElectrodeComponents

Primary hippocampal neurons are grown under highly standardizedconditions described above. The following cells are used: Young ControlNeurons (“YC”) isolated from fresh cadavers of adolescents or youngadults (ages 15 to 30 years); Age-matched Control Neurons (“AC”)isolated from fresh cadavers of elderly humans (60 to 80 years old); andAlzheimer's Disease Neurons (“AD”) isolated from fresh cadavers ofelderly humans with clinically diagnosed Alzheimer's Disease (60 to 80years old). Neuroblastoma×glioma hybrids (eg. the NG-108-15 cell line)and cells transfected with the gene for amyloid precursor protein areequally useful.

Cells are seeded (approximately 5 cells per mm²) onto microelectrodearrays, prepared as described above, in enriched culture medium and usedwhen impedance measurements indicate establishment of a high impedanceseal between at least one neuron and the substratum.

Electrophysiological measurements are performed at room temperature(21-23° C.) or, in other cases, as a function of temperature. Beforerecordings, culture medium is replaced with the following solution: 115mM NaCl, 40 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES (NaCl) pH=7.4,or in the alternative, fresh medium with selected neurotransmitter.Records are obtained using the instrumentation described below, linkedto a personal computer for storage and analysis.

Multiple types of sodium, calcium, potassium channels within each celland cell type are distinguished and recorded based on parameters of eachchannel, including conductance and current. Differences in ion channelproperties between cells of different lineage are also recorded, inother measurements, the response of cells to a battery ofneurotransmitter agonists is recorded, where the neurotransmittersinclude glutamate, carbachol, gamma amino butyric acid (GABA) andserotonin.

Specific but partial attenuation of ion channels with graded doses oftoxins including TEA (K⁺ channel), tetrodotoxin (Na⁺ channel) andamiloride (Ca²⁺ channel) are used in conjunction with deconvolution ofthe action potentials to determine the effect of model inhibitors.Additional agents and channel blockers are also used, including, but notlimited to: strychnine, Red Tide toxin, verapamil, nifedipine,ω-conotoxin, ω-agatoxin, apamin, quinine, charybdotoxin, dendrotoxin,maitoxin, and Ba²⁺.

64.6.7. TEA-Ca²⁺ Diagnostic Test

Primary neuronal cells are grown as described above. Thirteen AD, tenAC, and six YC are used for the calcium-flux and calcium imagingexperiments. Culture medium is replaced and washed three times withbasal salt solution (“BSS”) consisting of 140 mM NaCl, 5 mM KCl, 2.5 mMCaCl₂, 1.5 mM MgCl₂, 5 mM glucose, 10 mM HEPES (NaOH), pH 7.4. NominallyCa²⁺ free BSS is prepared as BSS without adding CaCl₂.

Fura-2 (acetyloxymethyl ester) (Fura-2AM) is purchased from MolecularProbes (Eugene, Oreg.) and stored as a 1 mM solution indimethylsulfoxide. Fura-2AM is added to a final concentration of 2 μMand cells are incubated at room temperature (21-23° C.) for 60 minutes.After incubation, cells are washed at least three times with BSS at roomtemperature before [Ca²⁺], determinations. Fluorescence is measured witha Hamamatsu ARGUS 50 imaging system (Hamamatsu Photonics, Japan) underthe control of a personal computer (Hamamatsu imaging software package).Excitation at 340 nm and 380 nm is attenuated with neutral densityfilters. Fluorescent images are obtained with a 400 nm dichroic mirrorand a 510 nm long-pass barrier filter. The objective lens is an X10Nikon UV fluor. Fluorescence is measured within a uniformly illuminatedfraction (¼) of the whole image.

The averaged Ca²⁺ responses within 15×15 pixels in cytosolic and innuclear cellular compartments obtained are quantified with ratiosbetween emitted 510 nm fluorescence activated at 340 nm and fluorescenceemitted at 510 nm with activation at 380 nm. These ratios aretransformed to absolute values of [Ca²⁺], after calibration based on thefollowing equation:

R=R _(max)+(R _(min) −R _(max))/(1+([Ca²⁺],/K_(d))_(b)).

Here R denotes fluorescence intensity illuminated by 340 nm divided byfluorescence intensity illuminated by 380 nm (F340/F380), and R_(max)and R_(min) are the values of R when the concentration of calcium is ata maximum and a minimum (i.e., the maximum and minimum value measurableby the machine under the measuring conditions), respectively. K_(d) is adissociation constant of fura-2 for Ca²⁺ and is determined at 240 nM.The value of b, which depends on the degree of asymmetry, is typically1.2. TGA application is used to cause a minimum of 100% [Ca⁺²],elevation in at least 18% of cells in every control cell. A response of100% [Ca⁺²], elevation in at least 10% of cells in a line is, therefore,a conservative criterion for a positive response.

The measurements of free intracellular calcium ion concentration([Ca²⁺],) is correlated to the Ca²⁺ flux as measured by the deconvolvedaction potential, using specifically the current corresponding tocalcium ion flux.

The neurons are depolarized by infusion of elevated external potassiumin order to distinguish the elevation of intracellular Ca²⁺ ([Ca²⁺],) inYC compared to AC and AD neurons. The depolarization-induced [Ca²⁺],elevation is eliminated by decreasing external calcium or by addingcalcium channel blockers. The neurons are depolarization by addition ofhigh K⁺ to cause a marked [Ca²⁺,] elevation in the various cell groups.The duration of the spike train of the calcium channel is correlated tothe [Ca²⁺], peak as measured by FURA-2 fluorescence and can be blockedif external calcium is lowered by substitution of “nominally Ca²⁺ free”solution or 5 mM EGTA (estimated free Ca²⁺=0.04 μM) or Ca²⁺ channelblockers (0.1 mM LaCl₃, 10 mM CoCl₂, 10 mM NiCl₂, 10 mM CdCl₂ or 10 μMnifedipine) before stimulation.

The neurons are depolarized by addition of TEA to cause [Ca²⁺],elevation, which is eliminated by decreasing external calcium or byadding calcium channel blockers. The AD neurons, can show [Ca²⁺],elevation in elevated external potassium without [Ca²⁺], response withaddition of 100 mM TEA. Moreover, the AC and YC cells can respond toTEA, even when the AD cells do not.

The application of 1 mM TEA is used to elevate [Ca²⁺], in YC neurons.The application of 10 mM TEA is used to elevate [Ca²⁺], in YC and ACneurons. Similarly application of 100 mM TEA is used to elevate [Ca²⁺],in YC and AC neurons. Similarly, the absence of external calcium ion isused to reduce or eliminate the response.

TEA is used to induce [Ca²⁺], elevations for coded samples that includeAlzheimer's and control neurons. Measurements and analyses are conductedwithout the experimenter's knowledge of the cell identity. The resultsare recorded for comparison with the non-blind sample.

Alzheimer's neurons from familial and non-familial cases are furtherused to evaluate agents with possible ameliorative properties. Forexample, choline esterase inhibitors can be compared using the system ofthe invention. After evaluation of the action potentials in YC, AC, andAD neurons, including deconvolution of the waveforms and comparison withknown ion channel inhibitors, graded doses of choline esteraseinhibitors are added separately, including:3-[1-(phenylmethyl)-4-piperidinyl)-1-(2,3,4,5-tetrahydro-1H-1-benzazepin-8-yl)-1-propanonefumarate, metrifonate, donepezil, tacrine, and rivastigmine. Theresultant action potentials are analyzed by application of the systemalgorithm to determine absolute and relative effects on nerve functionand calcium channel function.

6.4.6.8. Bomhesin-Ca²⁺ Diagnostic Test

Human hippocampal neurons described above are used according to theculture methods and electrophysiological methods described above.Bombesin is purchased from Calbiochem (San Diego, Calif.) and stored asa 1 mM solution in distilled water. Fura-2 (acetyloxymethyl ester),fura-2 (pentapotassium salt) and ω-conotoxin (ω-CgTX) GVIA are fromMolecular Probes (Eugene, Oreg.). Fura-2 AM is stored as a 1 mM solutionin dimethylsulfoxide; fura-2 pentapotassium salt is stored as a 6 mMsolution in potassium acetate, and ω-CgTX is stored as a 100 μM solutionin distilled water. All of the chemicals except for phenyloin aremaintained at −20° C. and protected from light.

The cells are incubated with 2 μM fura-2 AM in BSS (described above) atroom temperature (21-23° C.) for 60 min. After being washed at leastthree times with BSS, the cells are used for measurement of [Ca²⁺], atroom temperature. Cell fluorescence is measured as described above.Absolute calcium values and calcium fluxes are calculated as shownabove.

Bombesin is added to the cells at a final concentration of 1 μM. Calciummobilization levels are measured from −30 seconds to 150 seconds afterbombesin treatment. The maximum difference in [Ca²⁺], between AD cellsand control cells is determined and correlated to the differences incalcium channel function.

Bombesin is used to stimulate IP₃-induced Ca²⁺ release fromintracellular storage sites in neurons from all groups and to cause alarger and more prolonged response in AD neurons. The relationship ofthis larger and prolonged response in AD cells to extracellular Ca²⁺ isdetermined. On the other hand, the IP₃-mediated Ca²⁺ responses in AC andYC cells are followed by Ca²⁺ entry and by the effects on the ionchannels. When this Ca²⁺ entry is diminished by removal of extracellularCa²⁺, or blocking with inorganic Ca²⁺ blockers, the bombesin-elicitedCa²⁺ responses in control cells are returned to the basal level fasterthan in AD cells. Thus, this test is used to independently confirm theassessment made by the test above, based on potassium channeldysfunction See U.S. Pat. No. 5,976,816.

6.4.6.9. Effect of Rivastigmine and Nimodipine in a Model forNeurodegenerative Disease Action Potential Modulation in NeuronsOverexpressing β-Amyloid

The system of the invention is used to evaluate the efficacy ofexperimental agents in ameliorating the consequences or causes ofneurodegenerative diseases. In particular, a model for Alzheimer'sDisease is constructed by inducing the overexpression of β-amyloid inneurons. Sec U.S. Pat. No. 6,037,521 the disclosure of which isincorporated herein by reference, in its entirety. Models for otherneurodegenerative diseases are known in the art and can equally well beused.

Hippocampal neurons transfected with the plasmids pfβ/NORβ, pβA/fADβ,pβA/Dβ, pβA/ΔNORβ and pβA/NLβ from U.S. Pat. No. 6,037,521 areseparately plated onto microelectrode arrays and the adherence monitoredby inverted optical microscopy and by an increase in the impedance ateach microelectrode. The medium is modified to mimic the cerebrospinalfluid of Alzheimer's patients. After anchorage of the cells anddevelopment of a high impedance seal, the response of the normal andtransfected cells to stimulation with non-specific neuronal stimulators,including KCl and a shift in pH, is measured. Moreover, the effects ofspecific neurotransmitters, including, but not limited to carbacol,glutamate, dopamine, norepinephrine, serotonin, and GABA are separatelyevaluated. The action potentials are deconvolved for the biologicalanalysis of individual neurons. Following the deconvolution analysis, afirst phase of system analysis (also known a manipulation algorithm) isdirected toward the effect of the disease-associated media conditionsand the transfection with disease-associated gene constructs. Agents ofinterest for the treatment of neurodegenerative disease are then appliedto the neurons on the array. Acetylcholine esterase inhibitors, calciumchannel blockers, and cellular redox inhibitors are of particularinterest for Alzheimer's Disease, but other agents can equally well beevaluated. A key strength of the invention is the ability to quicklyscreen a large number of a wide variety of experimental and clinicalagents. For example, control neuronal cells and neuronal cellstransfected with pfβ/NORβ, pβA/FADβ, pβA/Dβ, pβA/ΔNORβor pβA/NLβ areseparately treated with graded doses of rivastigmine, an acetylcholineesterase inhibitor, or nimodipine, a calcium channel blocker. Therelative effects of the agents on the deconvolved action potentialsprovides identification of the ion channel involvement as a function ofthe disease model, and are compared to a library of known responses toion channel agonists, antagonists, and toxins. Then the resultant dataare further analyzed in a second phase system algorithm (manipulationalgorithm) to elucidate the effect of pfβ/NORβ, pβA/FADβ, pβA/Dβ,pβA/ΔNORβ and pβA/NLβ on energy metabolism, amino acid biosynthesis,cellular processes, lipid metabolism, regulation of transcription, andtransport and binding proteins. The results are further quantified interms of the relative effects of the agents on these major cellularsystems. Thus, it is expected that some agents, although putativelyacting by similar molecular pathways, can have differential effects thatare reflected in the action potential and which correspond todifferences in clinical efficacy and potency.

6.4.6.10. Measurement of Cell Surface Receptor Activation andIntracellular Signaling Via Second-Messenger Responsive Elements

Activation of cell surface receptors leads to a change in intracellularmessenger concentrations which in turn modulates intracellulartranscription factor activity. In neurons, an increase the intracellularconcentration of the messenger ion calcium leads to the activation ofthe nuclear factors. An increase in calcium levels alone is sufficientto markedly increase transcription of a reporter gene such as6-lactamase and to modulate the action potential.

Rat hippocampal neurons anchored to microelectrode arrays aretransiently cotransfected in situ with two plasmids. One plasmidcontains the β-adrenergic receptor, which localizes at the cells'surface, under the transcriptional control of the strong andconstitutively active cytomegalovirus (CMV) promoter. The other plasmidcontains the bacterial RTEM β-lactamase gene from Escherichia colimodified for improved mammalian expression under the transcriptionalcontrol of a promoter containing a trimer of NFAT sites. The plasmidsare introduced into cells using electroporation. 5×10⁶ cells in 0.5 mlelectroporation buffer are electroporated in the presence of 10 μg eachof both plasmids using the Biorad Gene Pulser (250V, 960 μF, 16 μsec).Twenty-four hours after transfection, cells are incubated in thepresence or absence of the β-adrenergic agonist isoproterenol (10μmolar) and the stimulation continued for 5 hours until desensitizationof the response occurs. The action potentials resulting from stimulationwith isoproterenol and during the gradual desensitization are analyzedto determine the role of ion channels. The data manipulation, or systemalgorithm is then applied to evaluate the contribution of key cellregulation systems.

6.4.7. Stem Cell Culture and Use

CNS stem cells are the natural founder cells which are present in theprimordial and spinal cord structures during the normal fetaldevelopment. The CNS stem cell technology available from of a number ofcommercial companies including NeuralStem Biopharmaceuticals andBiowhittaker enables isolation, expansion, and differentiation of CNSstem cells in vitro. Thus, subpopulation of neurons found in vivo cannow be produced in vitro from their founding precursor cells. Thehallmarks of the technology are that the CNS stem cells can be isolatedin large numbers (>10⁷ cells per rat embryo brain), further expanded upto a billion-fold over a 30-day period in culture, and efficientlydifferentiated in vitro where up to 80% of the cells become neurons. Inaddition to their desirable property of stably maintaining intrinsicinformation through many cell divisions, the CNS stem cells are alsoremarkably plastic. Thus, exposure of the cells to single extracellularfactors can shift the fate specification of the cells largely into onecell type or another. For example, platelet-derived growth factor (PDGF)increases neuronal differentiation from 50% to 90% of the cells. Thushippocampal neurons and stem cells differentiated into neurons areuseful in the invention. Moreover, genes are transfected into theneurons and/or stem cells by standard methods to provide a cellularconstruct for analysis of exogenous gene function.

6.5. The Microelectrode-Neuron Interface

The methodology and rationale for creating the surface modificationprotocols for the placement of the neuronal cell bodies and the creationof the high impedance seal are described in this section.

Electrode arrays consist of solid substrates on which patterns ofconducting wires lead from the electrode sites to external connectingpads. Many compositions are suitable as substrate material. Thesubstrates are often glass, which is preferred for use with biological(inverted) microscopes, although silicon substrates are sometimes usedbecause of their compatibility with electronic processing and thepotential for on-site amplification of signals. The conductors (e.g.gold, indium tin oxide, polysilicon) are coated with an insulator,typically a plastic (e.g. polyimide, polysiloxane), or a glass (e.g.silicon dioxide and/or silicon nitride). Holes, typically 5 or 10 μm indiameter, are etched into the insulator to define the electrode site.The electrode sites are often inlaid with a desired interface material(e.g. gold, platinum, iridium oxide titanium nitride) to improverecording and biocompatibility. In a preferred embodiment, the surfaceof the insulation and electrodes are silicon dioxide and gold,respectively, which can each be further modified chemically asnecessary.

Metal microelectrodes are used as transducers for the signals generatedby the neurons or cardiac myocytes. The detection mechanism and how itwill affect circuit design is shown in FIG. 7. During the actionpotential, the Na⁺ and Ca²⁺ channels open and Na⁺ and Ca²⁺ ions aretransported, into the cell, across the membrane. Na⁺ and Ca²⁺ influxcauses membrane depolarization which, over the region of the cellularmembrane in contact with the metal microelectrode, results in acapacitive discharge current, I_(C)=C_(jm) d V_(m)/dt where C_(jm) isthe junction-membrane capacitance and V_(m) is the membrane potential.The fluctuation in charge polarization on the insulator over theinterface produces a voltage difference across the metal, which is thendetected as a change in the charging current of the microelectrode. Sometime later, the K⁺ channels open and K⁺ ions flow out of the cell,across the membrane, and a similar but opposite sign impulse is impartedto the electrode.

A suitable circuit model of the detector system is shown in FIG. 8. Thecircuit is essentially that shown by Fromherz et al. (Wheeler, B. C.(1999). “Real Time Techniques for Automatic Discrimination of SingleUnits”, book chapter, in press, to Methods for Neural EnsembleRecordings, M. Nicolelis (editor), CRC Press.) with the exception thatwe represent the electrode as an equivalent capacitance and the externalamplifier as an ideal, discrete device. The membrane voltage. V_(m), istaken to be an idealized AC source in parallel with the membranecapacitance, C_(m). Any voltage appearing across the membrane appearsacross this capacitance in parallel with the junction-membranecapacitance C_(jm). The signal current is divided between the sealresistance R_(seal) or coupling to the amplifier through the electrodecapacitance C_(elec). One embodiment of the instant invention is thehigh impedance seal prepared using surface chemistry and optimized tominimize the current through the seal. Alternatively, if R_(seal) isvery large, then the membrane voltage appears at the amplifier inputwith little attenuation; if it is small then a smaller amplitude signalproportional to the derivative of the signal appears at the amplifierinput.

The effect is illustrated by considering the voltage divisional theelectrode (for an ideal amplifier)

Vo/Vm=KR _(seal) /sqrt[R _(seal) ²+(1/jwC _(jm))²]

where K is the amplifier gain, and w is the radian frequency of thesignal component measured. For 10 μm square electrode pads fully coveredby a membrane with specific membrane capacitance of 1 μF/cm2, C_(jm) isapproximately 1 pF; at 1 kHz, its impedance is 0.16 Gohm. If the sealresistance is much larger than 0.16 Gohm, the output equals the membranepotential; if it is much smaller, the output is the derivative of themembrane potential. If the amplifier is non-ideal and the sealresistance is high, there will be capacitive voltage division, betweenC_(jm) (1 pF), C_(elect) (approximately 1 nF), and the amplifier inputcapacitance (3 pF for the preamp by Multichannel Systems). Approximately25% of the amplitude of the membrane voltage appears at the amplifier,while 75% appears across the membrane apposed to the electrode.

A major advantage of the system of the invention is dial it permitscollection of data from up to 32 neurons or other cells, all exposed toidentical conditions of temperature, culture medium, chemicalstimulators and inhibitors. Thus, data of very high statistical qualitycan be obtained. Moreover, the artisan skilled in the art will realizethat array can readily be prepared with more, or less, than 32electrodes, by, for example, changing the total size of the array or thespacing between electrodes.

The simple model shown here illustrates that the recordable signal willvary from one that is proportional to membrane potential to one that isproportional to its derivative. The variation is a function of the sealresistance, the membrane properties, and the amplifier circuitry. Thisobservation applies to both the preliminary work with patch electrodesas well as the patch quality coupling of the neurons to substrateelectrodes. This model can be further refined for the generation ofpatch recordings from cells tethered to electrodes.

6.5.1. Construction and Characterization of the Neuron-MicroelectrodeInterface

A key component of this invention is the electrical characteristics ofthe neuron electrode interface. The biological/silicon interface is theresult of the close contact between the glycocalyx of the neuron and thesilicon/modified silicon dioxide surface. The neuron is a special casein that not only are the adhesion and viability of the cell crucial, butthe ability to detect the electrical signals generated by the neuronwith the silicon-based device is critical. These signals arecapacitivity coupled to the surface so the signal falls as the distancebetween neuron membrane and electrode increases. These signals aredetected by capacitivity coupling to a metal electrode. A key element ofthe invention is the optimal interaction of the neuron with theinterface as well as detection of the signals in the solid state.

6.5.2. Construction of High Impedance Seals on Electrodes by SurfaceModification

The closer the neuronal membrane is to the metal microelectrode thestronger the signal that can be detected. Patch-clamp recording works sowell because a gigaohm seal is created between the surface of the celland the electrode tip using a combination of a pressure differential andsurface interactions. This gigaohm seal almost eliminates the flow ofions from the surrounding media and allows membrane potentials and evensmall amplitude channel activity to be monitored in response to stimuli.A gigaohm seal is the most desirable result but the minimum necessary isthat the noise is low enough compared to the signal to permit detailedanalysis of the waveform. In the present invention tight binding of theneuronal cell surface to a artificially created surface over themicroelectrodes is promoted to form a high resistance seal. A variety ofbiologically molecules that have strong interactions are used to createthe seal, including, but not limited to antibodies that are used asneuronal markers. Suitable antibodies include, but are not limited to,the following antibodies or their equivalents, which are available fromBD Biosciences.

TABLE 2 Neurobiology Antibodies Description Clone 14-3-3ε 12Acetylcholine Receptor α 26 Acetylcholine Receptor β 74Acetylcholinesterase 46 Adaptin α  8 Adaptin β 74 Adaptin γ 88 AF6 35Amphiphysin 15 AP180 34 ApoE 32 Arc 49 β1-Calcium Channel 44 B56α 23 Bad32 Bad 48 N-Cadherin 32 R-Cadherin 48 Clathrin Heavy Chain 23Connexin-43  2 Contactin 41 Dynamin 41 Dynamin II 27 GABA^(A) Receptor(α1 Subunit) Polyclonal GABA^(B) Receptor Polyclonal Glutamate Receptor(GluR1) Polyclonal Glutamate Receptor (GluR2 and GluR3) PolyclonalGlutamate Receptor (GluR2 and GluR4) 3A11 Glutamate Receptor (GluR2) 6C4Glutamate Receptor (GluR2) Polyclonal Glutamate Receptor (GluR4)Polyclonal Glutamate Receptor (GluR5, GluR6, GluR7) 4F5 GlutamateReceptor (mGluR1α) G209-488 Glutamate Receptor (MGluR1α) G209-2048 Adultand Embryonic N-CAM (140 and 180 kD Epitopes) 12F11 Adult and EmbryonicPan N-CAM N-CAM 13 Embryonic N-CAM 12F8 Neurite Cell Adhesion MoleculeL1-Related 5G3 NMDA (NR2A) Polyclonal NMDA (NR2B) Polyclonal NMDA (NR2C)Polyclonal NMDAR1 54.1 NMDAR1 (N1) Polyclonal NMDAR1 (C1) PolyclonalNMDAR1 (C2) Polyclona NMDAR1(C2′) Polyclonal (k) Opioid Receptor(N-terminus) Polyclonal (k) Opioid Receptor (N-terminus) KAB (μ) OpioidReceptor Polyclonal (μ) Opioid Receptor (N-terminus) Polyclonal (δ)Opioid Receptor Polyclonal Serotonin Receptor (5-HT2AR) G186-I117Serotonin Receptor (5-HT2BR) A72-1 Serotonin Receptor (5-HT2CR) A4-2

Similar antibodies are also readily available from other sources, or canbe prepared by standard methods. They are linked to the thiols on thegold via standard crosslinking chemistry. These results are monitored bymeasuring the impedance at the metal microelectrodes. The proteinbuildup from the cells on the interface is monitored using surfaceanalysis because excess buildup increases the distance between cell andmetal microelectrode and thereby decreases signal strength.

6.5.3. Alternative Surfaces

Other surfaces and modifications are useful in segregating the cells,maintaining long term pattern fidelity and developing high resistanceseals between cells and microelectrode chip. SAMs are used as templatesfor further derivatization by charge-induced condensations,heterobifunctional crosslinkers, or simple adsorption. In addition toantibodies, as above, oilier macromolecules including bovine scrumalbumin (USA), laminin, tenascin. Neural Cell Adhesion Molecule (NCAM),L1, basic Fibroblast Growth Factor (bFGF), and severalglycosaminoglycans (GAGs) are suitable to form a tight seal with cells.The rationales for selecting these particular molecules are as follows:

(i) BSA: Albumin is a particularly prevalent protein in developingsystems, and may play an important role in supporting thedifferentiation of embryonic cells. In addition, albumin has been shownto prevent the non-specific adsorption of other proteins. (Ligler, F.S., Calvert, J. M. Georger, J. H., Shriver-Lake, L. C, Bhatia, S. K.,Bredchorst, R. U.S. Pat. No. 5,077,210 (1991).)

(ii) Laminin, tenascin, and NCAM: Laminin encourages neuronal growth.NCAM may induce proliferation of neurons. Tenascin has been shown to berepulsive to neurons and glia, and may be useful in keeping cells off ofcertain regions of patterned surfaces.

(iii) Growth factors (bFGF, BDNF): Growth factors are used as signals tocells to develop or differentiate, but their exact function is notclear. bFGF and BDNF promote neuronal survival.

(iv) GAGs: These polysaccharides represent the other major class ofextracellular macromolecules (other than proteins) that make up the ECM.GAGs suitable for the invention include HS (heparan sulfate),chondroitin sulfate, and hyaluronic acid

6.5.4. Controlled Placement of Neurons on Metals Using Surface Chemistry

As described above, two different SAMs can segregate onto two differentsurfaces from the same solution. This result can also be achievedsequentially with certain materials and can, for example, be used todifferentially modify the metal electrodes. The Si₃N₄/SiO₂ insulator ismodified with a silane and then the Au microelectrode is modified with aSAM that promotes cell body adhesion or provides anchors for biologicalmaterials that will promote this interaction. The SAMs on Au aretypically thiols. There is an extensive literature on their interactionwith various electronic materials (Hickman, J. J., Ofer. D., Laibinis,P. E. Whitesides, G. M., and Wrighton, M. S. (1991). Molecularself-assembly of two-terminal, voltammetric microsensors with internalreferences. Science 252: 688) and on methods for further derivatization.

6.5.5. Impedance Measurements

Impedances are measured by the method shown in FIG. 4. (Jung, D. R.;Cuttino, D. S.; Pancrazio, J. J.; P. Manos, P.; Custer, T.; Sathanoori,R. S.; Aloi, L. E.; Coulombe, M G;. Czarnaski, M. A.; Bondholder, D. A.;Kovacs. G. T. A.; Stenger, D. A.; Hickman, J. J. (1998). Cell-basedsensor microelectrode array characterized by imaging x-ray photoelectronspectroscopy, scanning electron microscopy, impedance measurements, andextracellular recordings. J. Vac. Sci. Technol. A, 16(3), May/June,1183-88.) A 100 mV root mean squared (rms) signal is applied by aplatinum wire in a 0.9% saline bath containing the microelectrode array.A lock-in amplifier (Model 5210 EC&G Princeton Applied Research) is usedto monitor the resulting phase and amplitude of the potential at (hisfrequency (typically 1 kHz or 100 Hz) across a 1.01 M Ohm (precisionresistor between the microelectrode and ground, yielding the real andimaginary parts of the microelectrode impedance, Re(Z_(ma))=R_(ma) andIm(Z_(ma))=X_(ma)=−j/(C_(ma)).

6.5.6. Surface Characterization

6.5.5.1. Surface Characterization Prior to Cell Culture

To relate the morphological and electrical properties of culturedneurons to changes in the conditions, each SAM-modified surface ischaracterized before each use by contact angle analysis, and XPS, ifappropriate. Contact angle measurements are a way of quantitating thesurface free energy of a modified surface. Characterizing surfacehydrophobicity or hydrophilicity permits determination of relativehydrophobicity or functional group accessibility and is correlated tocell adhesion or phenotype XPS is a technique for the elemental analysisand characterization of the overlayers on surfaces that allowsdetermination of efficiency of the modification techniques and alsofailure modes for establishing a good seal.

6.5.5.2. Surface Characterization after Cell Culture

The role or the surface in supporting neuronal growth is determined bymeasuring any reproducible changes in the amount, thickness, ordistribution of macromolecules on the underlying SAMs after cell culturethe emphasis is on the quantification of surface properties usingquantitative XPS, imaging XPS, and biological assays as appropriate ornecessary.

6.6. Device Fabrication and Testing

Planar electrode array recording of cultured neurons and muscle cellshas been reported for nearly three decades since the work of Thomas etal. in 1972. The basic technology for recording is standard and wellunderstood and, at present, there is ongoing and significant improvementin the commercial offerings of both arrays and supporting electronics.The standard instrumentation and data recording system in the inventionis described here. Detailed descriptions of the commercially availableplatforms are well known to those skilled in the art.

The supporting electronic instrumentation consists of standardpreamplifiers, standard amplifiers, standard filters, and a standardcomputer data acquisition interface. The system is capable of amplifyingall channels simultaneously with adjustable gain and bandpass filteringso that signals in the range of a few microvolts to a few millivolts,and from a few Hz to 5000 Hz, can be recorded digitally. In addition,the system makes provision for either manual or entirely computercontrolled electrical stimulation at individual electrode sites,including artifact rejection.

Action potentials (APs or spikes) are the primary signals recordablefrom the neurons cultured over the electrode arrays, in agreement withthe experience of a number of investigators recording from cultured,dissociated neurons. (Wheeler, B C. & Brewer, G J 1994. Multineuronpatterning and recording. In McKenna & Stenger (Eds.), EnablingTechnologies for Cultured Neural Networks (pp. 167-185). AcademicPress.) In addition, there may be occasional subthreshold events, ininstances where there is excellent electrical coupling between neuronand electrode, and occasional field potential signals where the densityof neurons is very high. The data recording of the invention focusesprimarily on AP detection and analysis, while preserving the capabilityfor logging and analyzing other types of signals. This will beconsidered in the development of the deconvolution algorithms.

6.6.1. Commercial Recording Systems

At present Plexon Inc. Dallas Tex. provides a product suitable for theinvention capable of real-time sorting of spikes on 64 channels (and asmany as 128); the software is designed to include automated clustercutting as well as flexibility in the choice of spike sorting features.The system includes storage of detected waveforms to disk for purposesof reclassification of signals off-line. The product is supported by anexcellent data analysis software package (Neuroexplorer) directed atanalyzing spike train signals. The product line begins with connectorsto electrode arrays and includes preamplifier and filtering.

The 64 channel MNAP (Multichannel Neuronal Acquisition Processor)includes high end personal computer, head stage stimulation/recordingpreamplifiers, input signal conditioning (SIG boards), A/D conversion(ADC boards) and digital signal processing (DSP boards), high speed hostdata communication (DCC boards), and output monitor (OUT board).

Multichannel Systems (Reutlingen, Germany) provides a product capable ofcontinuous time acquisition of signals from 64 channels including alimited real-time ability to detect interesting events for subsequentdata analysis. The data analysis software suggest a powerful product.The system includes electrode arrays and a compact stage pre-amplifierwhich greatly simplify recording logistics.

6.7. Protocol for High Impedance Throughput Analysis

The input-output relationships (spectral density and phase) of theextracellular clamp are derived from applying subthreshold amplitudesinusoidal voltage-clamp waveforms and measuring the output. Incontrast, on a chip microelectrode, the ideal interface causes no signalattenuation or phase shift; however, the capacitive coupling of theinterface imposes a low-pass filtering characteristic. Thus, thespectral density and phase relations provide quantitative measures ofthe interface characteristics for comparison among various SAM orSAM-modified surfaces.

6.8. Connection of the Apparatus of the Invention to a Computer

The apparatus of the invention may advantageously be connected to acomputer or to a computer network.

One of ordinary skill in the art will recognize that with respect to theabove-described computer network, the scope of the instant inventionincludes any suitable internet (lower case), i.e., any set of networksinterconnected with devices, such as routers, that forward messages orfragments of messages between networks or intranets. Naturally, theInternet (upper case) is one of the largest examples of an internet.

To this end, it is to be understood that the elements of the serviceprovider network, can be located in geographic proximity to one anotherin a substantially centralized processing environment, or canalternatively be arranged in a standard distributed processingenvironment so as to leverage resources, e.g., servers and storagedevices, located at two or more sites.

In an alternative embodiment, the above-mentioned computer network mayinclude a virtual private network (VPN), thereby taking advantage ofexisting PSTN infrastructure while providing a secure and privateenvironment for information exchange regarding resource usage.Advantageously, data sent from the VPN is encrypted, thereby enhancingthe privacy of customers. That is, because the VPN includes a tunnelingprotocol, the instant invention effectively uses the Internet as part ofa private secure network. That is, the “tunnel” is the particular paththat a given company message or file might travel through the Internet.

In another embodiment, the above-described computer network mayalternatively include an extranet, wherein customers may securelyexchange large volumes of resource usage data using a standard dataexchange formal, for example. Electronic Data Interchange. To thisextent, an extranet may enable customers to share news of commoninterest, for example, aggregated resource usage, exclusively withpartner companies.

It should be understood dial although standard graphical user interfacebrowsers have been discussed, standard text-only browsers, such as Lynx,may be used for UNIX shell and VMS users. Users of such text-onlybrowsers may download comma-delimited ASCII files of, for example, theirusage data.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without deviating from the spiritor scope of the present invention. Hence, no limitations on the scope ofthe invention should be implied by the specific embodiments chosen toillustrate the invention.

1.-49. (canceled)
 50. A system for identifying one or more ion channelsof a cell that may be affected by a test substance by deconvoluting achange in cell membrane potential, comprising: (a) a solid statemicroelectrode array; (b) a serum-free cell culture comprising one ormore electrically active cells having a cell membrane including one ormore ion channels, which cells are capable of providing a measurableaction potential that exhibits changes in one more perceptablecharacteristics selected from after potential, time to cessation ofactivity, frequency, amplitude, shape, spike rate, or time constant inresponse to a test substance; (c) an intervening layer that is acting asa high impedance seal and which is positioned between saidmicroelectrode and said one or more cells of said cell culture, and (d)accompanying deconvolution software with instructions that can beimplemented by a computer to deconvolute changes in the action potentialof the cells upon exposure to the test substance, wherein thedeconvolution analysis does not include a spectral analysis that makesuse of a Fourier transformation.
 51. The system of claim 50, wherein theone or more characteristics exhibited by said action potential ismanifested in its waveform or a derivative thereof.
 52. The system ofclaim 51, in which the one or more characteristics include at least oneof time to cessation of activity, frequency, amplitude, or shape. 53.The system of claim 50, in which the instructions comprise dataprocessing instructions capable of receiving input data comprising dateon ion flux through ion channels selected from the group consisting ofsodium channels, potassium channels, calcium channels, and combinationsthereof.
 54. The system of claim 50, in which the microelectrode isplanar or flexible.
 55. The system of claim 50, in which themicroelectrode is a field effect transducer.
 56. The system of claim 50,which further comprises an insulator layer surrounding themicroelectrode selected from the group consisting of silicon, modifiedsilicon dioxide, silicon nitride, silicon carbide, germanium, silica,gallium, arsenide, epoxy resin, polystyrene, polysulfone, alumina,silicone, fluoropolymer, polyester, acrylic copolymers, polylactate, orcombinations thereof.
 57. The system of claim 50 in which saidelectrically active cell comprises a neuronal cell or a cardiac cell.58. The system of claim 57, in which the neuronal cell is a hippocampalcell.
 59. The system of claim 50, in which the cell culture comprises astem cell, a transformed stem cell, their respective progeny, or acombination thereof.
 60. The system of claim 50, in which saidintervening layer comprises a self-assembling monolayer or monolayers.61. The system of claim 60, in which the self-assembling monolayercomprises a silane, a thiol, isocyanide, polyelectrolyte or combinationsthereof.
 62. The system of claim 50, wherein the intervening layerfurther comprises cell anchorage molecules selected from the groupconsisting of antibodies, antigens, receptor ligands, receptors,lectins, carbohydrates, enzymes, enzyme inhibitors, biotin, avidin,streptavidin, RGD-type peptides, integrins, cadherins, modified lipids,and combinations thereof.
 63. The system of claim 50, wherein theintervening layer further comprises a high viscosity mixture comprisingalcohols, ethers, esters, ketones, amides, glycols, amino acids,saccharides, carboxymethylsaccharides, carboxyethylsaccharides,aminosaccharides, acylaminosaccharides, polymers thereof, orcombinations thereof.
 64. The system of claim 50, in which one or morecells are transfected with endogenous or exogenous nucleic acid.
 65. Thesystem of claim 64, in which the nucleic acid comprises a nucleotidesequence associated with known function.
 66. The system of claim 50,wherein the cell culture is coated with a polymer.
 67. The system ofclaim 66, in which the polymer comprises cellulose, methylcellulose, ordextran.
 68. The system of claim 50, wherein a second layer is incontact with the electrically active cells and is attractive to celladherence.
 69. The system of claim 50, in which the test substancecomprises a toxin, a drug, a pathogen, a neurotransmitter, a nerveagent, or mixtures thereof.
 70. The system of claim 50, in which thedeconvolution of cell membrane potential includes deconvoluting the cellaction potential or its derivative.
 71. The system of claim 50, whereininformation on pathways in the cell is derived using a data library ofknown compounds classified into one or more functional categories. 72.The system of claim 71, wherein said functional categories arephosphatidylinositol turn-over, calcium mobilization, phosphorylation ofintracellular protein messengers, ion channel activators, ion channelblockers, transport proteins, binding proteins, cAMP formation, cellenvelope and membrane function, cell regulatory functions, amino acidbiosynthesis, fatty acid metabolism, phospholipid metabolism, steroidmetabolism, glycolysis, cellular maintenance processes, gene expression,neurotransmission inhibitors, protein synthesis inhibitors, energymetabolism, transcription, translation, G-protein coupled receptorfunction, or receptor transduction.
 73. The system of claim 64, in whichthe nucleic acid comprises a nucleotide sequence associated with unknownfunction.
 74. A computer-readable medium encoding a program withinstructions for execution by a computer in a system for identifying oneor more ion channels of a cell that may be affected by a test substanceby deconvoluting a change in cell membrane potential, said systemcomprising: (a) a solid state microelectrode array; (b) a serum-freecell culture comprising one or more electrically active cells having acell membrane including one or more ion channels, which cells arecapable of providing a measurable action potential that exhibits changesin one more perceptable characteristics selected from after potential,time to cessation of activity, frequency, amplitude, shape, spike rate,or time constant in response to a test substance; (c) an interveninglayer that is acting as a high impedance seal and which is positionedbetween said microelectrode and said one or more cells of said cellculture, and (d) accompanying deconvolution software with instructionsthat can be implemented by a computer to deconvolute changes in theaction potential of the cells upon exposure to the test substance,wherein the deconvolution analysis does not include a spectral analysisthat makes use of a Fourier transformation.
 75. The computer-readablemedium of claim 74, wherein information on pathways in the cell isderived using a data library of known compounds classified into one ormore functional categories.
 76. The computer-readable medium of claim75, wherein said functional categories are phosphatidylinositolturn-over, calcium mobilization, phosphorylation of intracellularprotein messengers, ion channel activators, ion channel blockers,transport proteins, binding proteins, cAMP formation, cell envelope andmembrane function, cell regulatory functions, amino acid biosynthesis,fatty acid metabolism, phospholipid metabolism, steroid metabolism,glycolysis, cellular maintenance processes, gene expression,neurotransmission inhibitors, protein synthesis inhibitors, energymetabolism, transcription, translation, G-protein coupled receptorfunction, or receptor transduction.
 77. The computer-readable medium ofclaim 74, in which one or more cells are transfected with endogenous orexogenous nucleic acid.
 78. The computer-readable medium of claim 77, inwhich the nucleic acid comprises a nucleotide sequence associated withknown function.